Novel Thiophene Inhibitors of S-Nitrosoglutathione Reductase

ABSTRACT

The present invention is directed to novel thiophene inhibitors of S-nitrosoglutathione reductase (GSNOR), pharmaceutical compositions comprising such GSNOR inhibitors, and methods of making and using the same.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/513,864, filed Jun. 5, 2012, entitled “Novel Thiophene Inhibitors ofS-Nitrosoglutathione Reductase”, now U.S. Pat. No. 8,586,624, whichapplication is a 35 U.S.C. §371 national phase application ofPCT/US2010/060303, filed Dec. 14, 2010 (WO 2011/075478).PCT/US2010/060303 claims the benefit of U.S. Provisional ApplicationSer. No. 61/286,881, filed Dec. 16, 2009. Each of the above-listedapplications is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to novel thiophene compounds,pharmaceutical compositions comprising such compounds, and methods ofmaking and using the same. These compounds are useful as inhibitors ofS-nitrosoglutathione reductase (GSNOR).

BACKGROUND

The chemical compound nitric oxide is a gas with chemical formula NO. NOis one of the few gaseous signaling molecules known in biologicalsystems, and plays an important role in controlling various biologicalevents. For example, the endothelium uses NO to signal surroundingsmooth muscle in the walls of arterioles to relax, resulting invasodilation and increased blood flow to hypoxic tissues. NO is alsoinvolved in regulating smooth muscle proliferation, platelet function,and neurotransmission, and plays a role in host defense. Although NO ishighly reactive and has a lifetime of a few seconds, it can both diffusefreely across membranes and bind to many molecular targets. Theseattributes make NO an ideal signaling molecule capable of controllingbiological events between adjacent cells and within cells.

NO is a free radical gas, which makes it reactive and unstable, thus NOis short lived in vivo, having a half life of 3-5 seconds underphysiologic conditions. In the presence of oxygen, NO can combine withthiols to generate a biologically important class of stable NO adductscalled S-nitrosothiols (SNO's). This stable pool of NO has beenpostulated to act as a source of bioactive NO and as such appears to becritically important in health and disease, given the centrality of NOin cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA,89:7674-7677 (1992)). Protein SNO's play broad roles in the function ofcardiovascular, respiratory, metabolic, gastrointestinal, immune, andcentral nervous system (Foster et al., Trends in Molecular Medicine, 9(4):160-168, (2003). One of the most studied SNO's in biological systemsis S-nitrosoglutathione (GSNO) (Gaston et al., Proc. Natl. Acad. Sci.USA 90:10957-10961 (1993)), an emerging key regulator in NO signalingsince it is an efficient trans-nitrosating agent and appears to maintainan equilibrium with other S-nitrosated proteins (Liu et al., Nature,410:490-494 (2001)) within cells. Given this pivotal position in theNO—SNO continuum, GSNO provides a therapeutically promising target toconsider when NO modulation is pharmacologically warranted.

In light of this understanding of GSNO as a key regulator of NOhomeostasis and cellular SNO levels, studies have focused on examiningendogenous production of GSNO and SNO proteins, which occurs downstreamfrom the production of the NO radical by the nitric oxide synthetase(NOS) enzymes. More recently there has been an increasing understandingof enzymatic catabolism of GSNO which has an important role in governingavailable concentrations of GSNO and consequently available NO andSNO's.

Central to this understanding of GSNO catabolism, researchers haverecently identified a highly conserved S-nitrosoglutathione reductase(GSNOR) (Jensen et al., Biochem J., 331:659-668 (1998); Liu et al.,(2001)). GSNOR is also known as glutathione-dependent formaldehydedehydrogenase (GSH-FDH), alcohol dehydrogenase 3 (ADH-3) (Uotila andKoivusalo, Coenzymes and Cofactors., D. Dolphin, ed. pp. 517-551 (NewYork, John Wiley & Sons, (1989)), and alcohol dehydrogenase 5 (ADH-5).Importantly GSNOR shows greater activity toward GSNO than othersubstrates (Jensen et al., 1998; Liu et al., 2001) and appears tomediate important protein and peptide denitrosating activity inbacteria, plants, and animals. GSNOR appears to be the majorGSNO-metabolizing enzyme in eukaryotes (Liu et al., 2001). Thus, GSNOcan accumulate in biological compartments where GSNOR activity is low orabsent (e.g., airway lining fluid) (Gaston et al., 1993).

Yeast deficient in GSNOR accumulate S-nitrosylated proteins which arenot substrates of the enzyme, which is strongly suggestive that GSNOexists in equilibrium with SNO-proteins (Liu et al., 2001). Preciseenzymatic control over ambient levels of GSNO and thus SNO-proteinsraises the possibility that GSNO/GSNOR may play roles across a host ofphysiological and pathological functions including protection againstnitrosative stress wherein NO is produced in excess of physiologicneeds. Indeed, GSNO specifically has been implicated in physiologicprocesses ranging from the drive to breathe (Lipton et al., Nature,413:171-174 (2001)) to regulation of the cystic fibrosis transmembraneregulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001)),to regulation of vascular tone, thrombosis, and platelet function (deBelder et al., Cardiovasc Res.; 28(5):691-4 (1994)); Z. Kaposzta, etal., Circulation; 106(24): 3057-3062, (2002)) as well as host defense(de Jesus-Berrios et al., Curr. Biol., 13:1963-1968 (2003)). Otherstudies have found that GSNOR protects yeast cells against nitrosativestress both in vitro (Liu et al., 2001) and in vivo (de Jesus-Berrios etal., (2003)).

Collectively, data suggest GSNO as a primary physiological ligand forthe enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizesGSNO and consequently reduces available SNO's and NO in biologicalsystems (Liu et al., (2001)), (Liu et al., Cell, 617-628 (2004)), and(Que et al., Science, 308, (5728):1618-1621 (2005)). As such, thisenzyme plays a central role in regulating local and systemic bioactiveNO. Since perturbations in NO bioavailability has been linked to thepathogenesis of numerous disease states, including hypertension,atherosclerosis, thrombosis, asthma, gastrointestinal disorders,inflammation, and cancer, agents that regulate GSNOR activity arecandidate therapeutic agents for treating diseases associated with NOimbalance.

Nitric oxide (NO), S-nitrosoglutathione (GSNO), and S-nitrosoglutathionereductase (GSNOR) regulate normal lung physiology and contribute to lungpathophysiology. Under normal conditions, NO and GSNO maintain normallung physiology and function via their anti-inflammatory andbronchodilatory actions. Lowered levels of these mediators in pulmonarydiseases such as asthma, chronic obstructive pulmonary disease (COPD)may occur via up-regulation of GSNOR enzyme activity. These loweredlevels of NO and GSNO, and thus lowered anti-inflammatory capabilities,are key events that contribute to pulmonary diseases and which canpotentially be reversed via GSNOR inhibition.

Inflammatory bowel diseases (IBD's), including Crohn's and ulcerativecolitis, are chronic inflammatory disorders of the gastrointestinal (GI)tract, in which NO, GSNO, and GSNOR can exert influences. Under normalconditions, NO and GSNO function to maintain normal intestinalphysiology via anti-inflammatory actions and maintenance of theintestinal epithelial cell barrier. In IBD, reduced levels of GSNO andNO are evident and likely occur via up-regulation of GSNOR activity. Thelowered levels of these mediators contribute to the pathophysiology ofIBD via disruption of the epithelial barrier via dysregulation ofproteins involved in maintaining epithelial tight junctions. Thisepithelial barrier dysfunction, with the ensuing entry ofmicro-organisms from the lumen, and the overall loweredanti-inflammatory capabilities in the presence of lowered NO and GSNO,are key events in IBD progression that can be potentially influenced bytargeting GSNOR.

Currently, there is a great need in the art for diagnostics,prophylaxis, ameliorations, and treatments for medical conditionsrelating to increased NO synthesis and/or increased NO bioactivity. Inaddition, there is a significant need for novel compounds, compositions,and methods for preventing, ameliorating, or reversing otherNO-associated disorders. The present invention satisfies these needs.

SUMMARY

The present invention provides novel thiophene compounds. Thesecompounds are useful as S-nitrosoglutathione reductase (“GSNOR”)inhibitors. The invention encompasses pharmaceutically acceptable salts,stereoisomers, prodrugs, and metabolites of the described compounds.Also encompassed by the invention are pharmaceutical compositionscomprising at least one compound of the invention and at least onepharmaceutically acceptable carrier.

The compositions of the present invention can be prepared in anysuitable pharmaceutically acceptable dosage form.

The present invention provides a method for inhibiting GSNOR in asubject in need thereof. Such a method comprises administering atherapeutically effective amount of a pharmaceutical compositioncomprising at least one GSNOR inhibitor or a pharmaceutically acceptablesalt, prodrug, stereoisomer, or metabolite thereof, in combination withat least one pharmaceutically acceptable carrier. The GSNOR inhibitorcan be a novel compound according to the invention, or it can be a knowncompound which previously was not known to be an inhibitor of GSNOR.

The present invention also provides a method of treating a disorderameliorated by NO donor therapy in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt, stereoisomer, prodrug, or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The present invention also provides a method of treating a cellproliferative disorder in a subject in need thereof. Such a methodcomprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt, stereoisomer, prodrug or metabolitethereof, in combination with at least one pharmaceutically acceptablecarrier. The GSNOR inhibitor can be a novel compound according to theinvention, or it can be a known compound which previously was not knownto be an inhibitor of GSNOR.

The methods of the invention encompass administration with one or moresecondary active agents. Such administration can be sequential or in acombination composition.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publiclyavailable publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control.

Both the foregoing summary and the following detailed description areexemplary and explanatory and are intended to provide further details ofthe compositions and methods as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description.

DETAILED DESCRIPTION A. Overview of the Invention

Until recently, S-nitrosoglutathione reductase (GSNOR) was known tooxidize the formaldehyde glutathione adduct, S-hydroxymethylglutathione.GSNOR has since been identified in a variety of bacteria, yeasts,plants, and animals and is well conserved. The proteins from E. coli, S.cerevisiae and mouse macrophages share over 60% amino acid sequenceidentity. GSNOR activity (i.e., decomposition of GSNO when NADH ispresent as a required cofactor) has been detected in E. coli, in mousemacrophages, in mouse endothelial cells, in mouse smooth muscle cells,in yeasts, and in human HeLa, epithelial, and monocyte cells. HumanGSNOR nucleotide and amino acid sequence information can be obtainedfrom the National Center for Biotechnology Information (NCBI) databasesunder Accession Nos. M29872, NM_(—)000671. Mouse GSNOR nucleotide andamino acid sequence information can be obtained from NCBI databasesunder Accession Nos. NM_(—)007410. In the nucleotide sequence, the startsite and stop site are underlined. CDS designates coding sequence. SNPdesignates single nucleotide polymorphism. Other related GSNORnucleotide and amino acid sequences, including those of other species,can be found in U.S. Patent Application 2005/0014697.

In accord with the present invention, GSNOR has been shown to functionin vivo and in vitro to metabolize S-nitrosoglutathione (GSNO) andprotein S-nitrosothiols (SNOs) to modulate NO bioactivity, bycontrolling the intracellular levels of low mass NO donor compounds andpreventing protein nitrosylation from reaching toxic levels.

Based on this, it follows that inhibition of this enzyme potentiatesbioactivity in diseases in which NO donor therapy is indicated, inhibitsthe proliferation of pathologically proliferating cells, and increasesNO bioactivity in diseases where this is beneficial.

The present invention provides pharmaceutical agents that are potentinhibitors of GSNOR. In particular, provided are substituted thiopheneanalogs that are inhibitors of GSNOR having the structure depicted below(Formula I), or a pharmaceutically acceptable salt, stereoisomer,prodrug, or metabolite thereof.

wherein:X and Y are selected from the group consisting of S or CH, provided thatwhen X is S, Y must be CH and when X is CH, Y must be S;Ar is selected from the group consisting of phenyl and thiophen-yl;R₁ is selected from the group consisting of hydrogen, unsubstitutedimidazolyl, substituted imidazolyl, carbamoyl, chloro, bromo, fluoro,hydroxy, and methoxy;R₂ is selected from the group consisting of hydrogen, methyl, chloro,fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl, dimethylamino,amino, formamido, and trifluoromethyl;R₃ is selected from the group consisting of hydrogen, methyl, methoxy,chloro, and fluoro; andR₄ is selected from the group consisting of CONH₂, NHSO₂CH₃, hydroxy,chloro, and substituted and unsubstituted imidazolyl.

As used in this context, the term “analog” refers to a compound havingsimilar chemical structure and function as compounds of Formula I thatretains the thiophene ring.

Some thiophene analogs of the invention can also exist in variousisomeric forms, including configurational, geometric, and conformationalisomers, as well as existing in various tautomeric forms, particularlythose that differ in the point of attachment of a hydrogen atom. As usedherein, the term “isomer” is intended to encompass all isomeric forms ofa compound including tautomeric forms of the compound.

Illustrative compounds having asymmetric centers can exist in differentenantiomeric and diastereomeric forms. A compound can exist in the formof an optical isomer or a diastereomer. Accordingly, the inventionencompasses compounds in the forms of their optical isomers,diastereomers and mixtures thereof, including racemic mixtures.

It should be noted that if there is a discrepancy between a depictedstructure and a name given to that structure, the depicted structurecontrols. In addition, if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold, wedged,or dashed lines, the structure or portion of the structure is to beinterpreted as encompassing all stereoisomers of the described compound.

B. S-Nitrosoglutathione Reductase Inhibitors

1. Inventive Compounds

In one of its aspects the present invention provides a compound having astructure shown in Formula I, or a pharmaceutically acceptable salt,stereoisomer, prodrug or metabolite thereof:

wherein:X and Y are selected from the group consisting of S or CH, provided thatwhen X is S, Y must be CH and when X is CH, Y must be S;Ar is selected from the group consisting of phenyl and thiophen-yl;R₁ is selected from the group consisting of hydrogen, unsubstitutedimidazolyl, substituted imidazolyl, carbamoyl, chloro, bromo, fluoro,hydroxy, and methoxy;R₂ is selected from the group consisting of hydrogen, methyl, chloro,fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl, dimethylamino,amino, formamido, and trifluoromethyl; andR₃ is selected from the group consisting of hydrogen, methyl, methoxy,chloro, and fluoro; andR₄ is selected from the group consisting of CONH₂, NHSO₂CH₃, hydroxy,chloro, and substituted and unsubstituted imidazolyl.

In a further aspect of the invention, Ar is selected from the groupconsisting of

wherein R₁ and R₂ are as defined previously.

In a further aspect of the invention, R₁ is

wherein R₅ is selected from the group consisting of hydrogen, methyl,and ethyl.

In a further aspect of the invention, ArR₁R₂ is selected from the groupconsisting of: 4-methoxyphenyl, 4-chloro-2-methoxyphenyl,4-hydroxyphenyl, 4-carbamoylphenyl, and 4-bromophenyl.

In a further aspect of the invention, R₃ is selected from the groupconsisting of hydrogen, methyl, and methoxy.

In one of its aspects the present invention provides a compound having astructure shown in Formula II, or a pharmaceutically acceptable salt,stereoisomer, prodrug or metabolite thereof:

wherein:Ar is selected from the group consisting of phenyl and thiophen-yl;R₁ is selected from the group consisting of hydrogen, unsubstitutedimidazolyl, substituted imidazolyl, carbamoyl, chloro, bromo, fluoro,hydroxy, and methoxy;R₂ is selected from the group consisting of hydrogen, methyl, chloro,fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl, dimethylamino,amino, formamido, and trifluoromethyl; andR₃ is selected from the group consisting of hydrogen, methyl, methoxy,chloro, and fluoro; andR₄ is selected from the group consisting of CONH₂, NHSO₂CH₃, hydroxy,chloro, and substituted and unsubstituted imidazolyl.

In a further aspect of the invention, Ar is selected from the groupconsisting of

wherein R₁ and R₂ are as defined previously.

In a further aspect of the invention, R₁ is

wherein R₅ is selected from the group consisting of hydrogen, methyl,and ethyl.

In a further aspect of the invention, ArR₁R₂ is selected from the groupconsisting of: 4-methoxyphenyl, 4-chloro-2-methoxyphenyl,4-hydroxyphenyl, 4-carbamoylphenyl, and 4-bromophenyl.

In a further aspect of the invention, R₃ is selected from the groupconsisting of hydrogen, methyl, and methoxy.

In a further aspect of the invention, suitable compounds of Formula IIinclude, but are not limited to:

-   3-(3-(4-carbamoyl-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)propanoic    acid;-   3-(3-(4-carbamoylphenyl)-4-(4-chloro-2-methoxyphenyl)thiophen-2-yl)propanoic    acid;-   3-(3-(4-chloro-2-methoxyphenyl)-4-(4-hydroxyphenyl)thiophen-2-yl)propanoic    acid;-   3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoic    acid;-   3-(4-(4-chloro-2-methoxyphenyl)-3-(4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoic    acid;-   3-(4-(4-carbamoylphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoic    acid;-   3-(3-(4-carbamoyl-2-methylphenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoic    acid;-   3-(3-(4-carbamoylphenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoic    acid;-   3-(4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-3-(2-methyl-4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoic    acid;-   3-(4-(4-chloro-2-methoxyphenyl)-3-(2-methyl-4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoic    acid;-   3-(4′-(4-carbamoyl-2-methylphenyl)-5-(2-methyl-1H-imidazol-1-yl)-2,3′-bithiophen-5′-yl)propanoic    acid;-   3-(5-(2-methyl-1H-imidazol-1-yl)-4′-(2-methyl-4-(methylsulfonamido)phenyl)-2,3′-bithiophen-5′-yl)propanoic    acid; and-   3-(4-(4-bromophenyl)-3-(4-carbamoyl-2-methylphenyl)thiophen-2-yl)propanoic    acid.

In one of its aspects the present invention provides a compound having astructure shown in Formula III, or a pharmaceutically acceptable salt,stereoisomer, prodrug, or metabolite thereof:

wherein:Ar is selected from the group consisting of phenyl and thiophen-yl;R₁ is selected from the group consisting of hydrogen, unsubstitutedimidazolyl, substituted imidazolyl, carbamoyl, chloro, bromo, fluoro,hydroxy, and methoxy;R₂ is selected from the group consisting of hydrogen, methyl, chloro,fluoro, hydroxy, methoxy, ethoxy, propoxy, carbamoyl, dimethylamino,amino, formamido, and trifluoromethyl; andR₃ is selected from the group consisting of hydrogen, methyl, methoxy,chloro, and fluoro; andR₄ is selected from the group consisting of CONH₂, NHSO₂CH₃, hydroxy,chloro, and substituted and unsubstituted imidazolyl.

In a further aspect of the invention, Ar is selected from the groupconsisting of

wherein R₁ and R₂ are as defined previously.

In a further aspect of the invention, R₁ is

wherein R₅ is selected from the group consisting of hydrogen, methyl,and ethyl.

In a further aspect of the invention, ArR₁R₂ is selected from the groupconsisting of: 4-methoxyphenyl, 4-chloro-2-methoxyphenyl,4-hydroxyphenyl, 4-carbamoylphenyl, and 4-bromophenyl.

In a further aspect of the invention, R₃ is selected from the groupconsisting of hydrogen, methyl, and methoxy.

In a further aspect of the invention, a suitable compound of Formula IIIincludes, but is not limited to:

-   3-(4-(4-carbamoyl-2-methylphenyl)-5-(4-methoxyphenyl)thiophen-3-yl)propanoic    acid.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Alltautomers of shown or described compounds are also considered to be partof the present invention.

It is to be understood that isomers arising from such asymmetry (e.g.,all enantiomers and diastereomers) are included within the scope of theinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Alkenes can include either the E- or Z-geometry,where appropriate.

2. Representative Compounds

Examples 1-14 list representative novel thiophene analogs of Formula I.The synthetic methods that can be used to prepare each compound aredetailed in Examples 1-14, with reference to synthetic schemes depictedbefore Example 1, and reference to intermediates described in Example15. Supporting mass spectrometry data and proton NMR data for eachcompound is also included in Examples 1-14. GSNOR inhibitor activity wasdetermined by the assay described in Example 16 and IC₅₀ values wereobtained. GSNOR inhibitor compounds in Examples 1-14 had an IC₅₀ ofabout <10 μM. GSNOR inhibitor compounds in Examples 1, 2, 4, 7-9, 11-13,14 had an IC₅₀ of about less than 1.0 μM.

C. Definitions

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

The term “acyl” includes compounds and moieties that contain the acetylradical (CH₃CO—) or a carbonyl group to which a straight or branchedchain lower alkyl residue is attached.

The term “alkyl” as used herein refers to a straight or branched chain,saturated hydrocarbon having the indicated number of carbon atoms. Forexample, (C₁-C₆) alkyl is meant to include, but is not limited tomethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. An alkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein.

The term “alkenyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one double bond. Examples of a (C₂-C₈) alkenyl group include,but are not limited to, ethylene, propylene, 1-butylene, 2-butylene,isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene,2-hexene, 3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene,isoheptene, 1-octene, 2-octene, 3-octene, 4-octene, and isooctene. Analkenyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkynyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one triple bond. Examples of a (C₂-C₈) alkynyl group include,but are not limited to, acetylene, propyne, 1-butyne, 2-butyne,1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne,2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. Analkynyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkoxy” as used herein refers to an —O-alkyl group having theindicated number of carbon atoms. For example, a (C₁-C₆) alkoxy groupincludes —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl,—O-sec-butyl, —O-tert-butyl, —O-pentyl, —O-isopentyl, —O-neopentyl,—O-hexyl, —O-isohexyl, and —O-neohexyl.

The term “aminoalkyl” as used herein, refers to an alkyl group(typically one to six carbon atoms) wherein one or more of the C₁-C₆alkyl group's hydrogen atoms is replaced with an amine of formula—N(R^(c))₂, wherein each occurrence of R^(c) is independently —H or(C₁-C₆) alkyl. Examples of aminoalkyl groups include, but are notlimited to, —CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂N(CH₃)₂,t-butylaminomethyl, isopropylaminomethyl, and the like.

The term “aryl” as used herein refers to a 5- to 14-membered monocyclic,bicyclic, or tricyclic aromatic ring system. Examples of an aryl groupinclude phenyl and naphthyl. An aryl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow. Examples of aryl groups include phenyl or aryl heterocycles suchas, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxy” or “carboxyl” means a —COOH group or carboxylic acid.

The term “C_(m)-C_(n)” means “m” number of carbon atoms to “n” number ofcarbon atoms. For example, the term “C₁-C₆” means one to six carbonatoms (C₁, C₂, C₃, C₄, C₅, or C₆). The term “C₂-C₆” includes two to sixcarbon atoms (C₂, C₃, C₄, C₅, or C₆). The term “C₃-C₆” includes three tosix carbon atoms (C₃, C₄, C₅, or C₆).

The term “cycloalkyl” as used herein refers to a 3- to 14-memberedsaturated or unsaturated non-aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system. Included in this class are cycloalkyl groupswhich are fused to a benzene ring. Representative cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl,cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, cycloheptyl, cycloheptenyl, 1,3-cycloheptadienyl,1,4-cycloheptadienyl, -1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctenyl, 1,3-cyclooctadienyl, 1,4-cyclooctadienyl,-1,3,5-cyclooctatrienyl, decahydronaphthalene, octahydronaphthalene,hexahydronaphthalene, octahydroindene, hexahydroindene, tetrahydroinden,decahydrobenzocycloheptene, octahydrobenzocycloheptene,hexahydrobenzocycloheptene, tetrahydrobenzocyclopheptene,dodecahydroheptalene, decahydroheptalene, octahydroheptalene,hexahydroheptalene, tetrahydroheptalene, (1s,3s)-bicyclo[1.1.0]butane,bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, Bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane,bicyclo[3.3.1]nonane, bicyclo[3.3.2]decane, bicyclo[3.3.]undecane,bicyclo[4.2.2]decane, and bicyclo[4.3.1]decane. A cycloalkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein below.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.

The term “haloalkyl,” as used herein, refers to a C₁-C₆ alkyl groupwherein from one or more of the C₁-C₆ alkyl group's hydrogen atom isreplaced with a halogen atom, which can be the same or different.Examples of haloalkyl groups include, but are not limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl,pentachloroethyl, and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chainalkyl, or combinations thereof, consisting of carbon atoms and from oneto three heteroatoms selected from the group consisting of O, N, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. The heteroatom(s)O, N, and S can be placed at any position of the heteroalkyl group.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, and—CH₂—CH═N—OCH₃. Up to two heteroatoms can be consecutive, for example,—CH₂—NH—OCH₃. When a prefix such as (C₂-C₈) is used to refer to aheteroalkyl group, the number of carbons (2 to 8, in this example) ismeant to include the heteroatoms as well. For example, a C₂-heteroalkylgroup is meant to include, for example, —CH₂OH (one carbon atom and oneheteroatom replacing a carbon atom) and —CH₂SH.

To further illustrate the definition of a heteroalkyl group, where theheteroatom is oxygen, a heteroalkyl group can be an oxyalkyl group. Forinstance, (C₂-C₅) oxyalkyl is meant to include, for example —CH₂—O—CH₃(a C₃-oxyalkyl group with two carbon atoms and one oxygen replacing acarbon atom), —CH₂CH₂CH₂CH₂OH, —OCH₂CH₂OCH₂CH₂OH, —OCH₂CH(OH)CH₂OH, andthe like.

The term “heteroaryl” as used herein refers to an aromatic heterocyclering of 5 to 14 members and having at least one heteroatom selected fromnitrogen, oxygen, and sulfur, and containing at least 1 carbon atom,including monocyclic, bicyclic, and tricyclic ring systems.Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl,pyridyl, furyl, benzofuranyl, thienyl, benzothienyl, quinolinyl,pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, quinoxalinyland oxazolyl. A heteroaryl group can be unsubstituted or optionallysubstituted with one or more substituents as described herein below.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), and sulfur (S).

As used herein, the term “heterocycle” refers to 3- to 14-membered ringsystems which are either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen, and sulfur, and wherein the nitrogen and sulfur heteroatoms canbe optionally oxidized, and the nitrogen heteroatom can be optionallyquaternized, including, including monocyclic, bicyclic, and tricyclicring systems. The bicyclic and tricyclic ring systems may encompass aheterocycle or heteroaryl fused to a benzene ring. The heterocycle canbe attached via any heteroatom or carbon atom, where chemicallyacceptable. Heterocycles include heteroaryls as defined above.Representative examples of heterocycles include, but are not limited to,aziridinyl, oxiranyl, thiiranyl, triazolyl, tetrazolyl, azirinyl,diaziridinyl, diazirinyl, oxaziridinyl, azetidinyl, azetidinonyl,oxetanyl, thietanyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl,oxazinyl, thiazinyl, diazinyl, dioxanyl, triazinyl, tetrazinyl,imidazolyl, tetrazolyl, pyrrolidinyl, isoxazolyl, furanyl, furazanyl,pyridinyl, oxazolyl, benzoxazolyl, benzisoxazolyl, thiazolyl,benzthiazolyl, thienyl, pyrazolyl, triazolyl, pyrimidinyl,benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl, benzotriazolyl,benzoxazolyl, benzisoxazolyl, purinyl, indolyl, isoquinolinyl,quinolinyl, and quinazolinyl. A heterocycle group can be unsubstitutedor optionally substituted with one or more substituents as describedherein below.

The term “heterocycloalkyl,” by itself or in combination with otherterms, represents, unless otherwise stated, cyclic versions of“heteroalkyl.” Additionally, a heteroatom can occupy the position atwhich the heterocycle is attached to the remainder of the molecule.Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like.

The term “hydroxyalkyl,” as used herein, refers to an alkyl group havingthe indicated number of carbon atoms wherein one or more of the hydrogenatoms in the alkyl group is replaced with an —OH group. Examples ofhydroxyalkyl groups include, but are not limited to, —CH₂OH, —CH₂CH₂OH,—CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂CH₂OH, and branched versions thereof.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein, N-oxide, or amine oxide, refers to a compound derivedfrom a tertiary amine by the attachment of one oxygen atom to thenitrogen atom, R₃N⁺—O⁻. By extension the term includes the analogousderivatives of primary and secondary amines.

As used herein and unless otherwise indicated, the term “stereoisomer”means one stereoisomer of a compound that is substantially free of otherstereoisomers of that compound. For example, a stereomerically purecompound having one chiral center will be substantially free of theopposite enantiomer of the compound. A stereomerically pure compoundhaving two chiral centers will be substantially free of otherdiastereomers of the compound. In some embodiments, a stereomericallypure compound comprises greater than about 80% by weight of onestereoisomer of the compound and less than about 20% by weight of otherstereoisomers of the compound, for example greater than about 90% byweight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment”. A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “modulate” is meant to refer to an increase or decreasein the levels of a peptide or a polypeptide, or to increase or decreasethe stability or activity of a peptide or a polypeptide. The term“inhibit” is meant to refer to a decrease in the levels of a peptide ora polypeptide or to a decrease in the stability or activity of a peptideor a polypeptide. In preferred embodiments, the peptide which ismodulated or inhibited is S-nitrosoglutathione (GSNO) or proteinS-nitrosothiols (SNOs).

As used here, the terms “nitric oxide” and “NO” encompass unchargednitric oxide and charged nitric oxide species, particularly includingnitrosonium ion (NO⁺) and nitroxyl ion (NO⁻). The reactive form ofnitric oxide can be provided by gaseous nitric oxide. Compounds havingthe structure X—NO_(y) wherein X is a nitric oxide releasing,delivering, or transferring moiety, including any and all such compoundswhich provide nitric oxide to its intended site of action in a formactive for their intended purpose, and Y is 1 or 2.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered and includes, but is not limitedto such sterile liquids as water and oils.

A “pharmaceutically acceptable salt” or “salt” of a compound of theinvention is a product of the disclosed compound that contains an ionicbond, and is typically produced by reacting the disclosed compound witheither an acid or a base, suitable for administering to a subject. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, and K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

A “pharmaceutical composition” is a formulation comprising the disclosedcompounds in a form suitable for administration to a subject. Apharmaceutical composition of the invention is preferably formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include, but are not limited to, oral andparenteral, e.g., intravenous, intradermal, subcutaneous, inhalation,topical, transdermal, transmucosal, and rectal administration.

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N, or N═N).

Substituents for the groups referred to as alkyl, heteroalkyl, alkylene,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl can be selected from a variety of groups including—OR^(d)′, ═O, ═NR^(d)′, ═N—OR^(d)′, —NR^(d)′R^(d)″, —SR^(d)′, -halo,—SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′, —C(O)R^(d)′, —CO₂R^(d)′,—CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)′″C(O)NR^(d)′R^(d)″, —NR^(d)′″SO₂NR^(d)′R^(d)″,—NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH, —NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′,—S(O)R^(d)′, —SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and—NO₂, in a number ranging from zero to three, with those groups havingzero, one or two substituents being exemplary.

R^(d)′, R^(d)″, and R^(d)′″ each independently refer to hydrogen,unsubstituted (C₁-C₈)alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, and aryl substituted with one to three substituentsselected from -halo, unsubstituted alkyl, unsubstituted alkoxy,unsubstituted thioalkoxy, and unsubstituted aryl (C₁-C₄)alkyl. WhenR^(d)′ and R^(d)″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR^(d)′R^(d)″ can represent 1-pyrrolidinyl or4-morpholinyl.

Typically, an alkyl or heteroalkyl group will have from zero to threesubstituents, with those groups having two or fewer substituents beingexemplary of the present invention. An alkyl or heteroalkyl radical canbe unsubstituted or monosubstituted. In some embodiments, an alkyl orheteroalkyl radical will be unsubstituted.

Exemplary substituents for the alkyl and heteroalkyl radicals include,but are not limited to —OR^(d)′, ═O, ═NR^(d)′, ═N—OR^(d)′,—NR^(d)′R^(d)″, —SR^(d)′, -halo, —SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′,—C(O)R^(d)′, —CO₂R^(d)′, —CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″,—NR^(d)′C(O)R^(d)′, —NR^(d)′″C(O)NR^(d)′R^(d)″,—NR^(d)′″SO₂NR^(d)′R^(d)″, —NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH,—NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′, —S(O)R^(d)′, —SO₂R^(d)′,—SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and —NO₂, where R^(d)′,R^(d)″, and R^(d)′″ are as defined above. Typical substituents can beselected from: —OR^(d)′, ═O, —NR^(d)′R^(d)″, -halo, —OC(O)R^(d)′,—CO₂R^(d)′, —C(O)NR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)″CO₂R^(d)′, —NR^(d)′″SO₂NR^(d)′R^(d)″, —SO₂R^(d)′,—SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: -halo, —OR^(e)′, —OC(O)R^(e)′, —NR^(e)′R^(e)″,—SR^(e)′, —R^(e)′, —CN, —NO₂, —CO₂R^(e)′, —C(O)NR^(e)′R^(e)″,—C(O)R^(e)′, —OC(O)NR^(e)′R^(e)″, —NR^(e)″C(O)R^(e)′, —NR^(e)″CO₂R^(e)′,—NR^(e)′″C(O)NR^(e)′R^(e)″, —NR^(e)′″SO₂NR^(e)′R^(e)″, —NHC(NH₂)═NH,—NR^(e)′C(NH₂)═NH, —NH—C(NH₂)═NR^(e)′, —S(O)R^(e)′, —SO₂R^(e)′,—SO₂NR^(e)′R^(e)″, —NR^(e)″SO₂R^(e)′, —N₃, —CH(Ph)₂, perfluoroalkoxy,and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system.

R^(e)′, R^(e)″ and R^(e)′″ are independently selected from hydrogen,unsubstituted (C₁-C₈) alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedaryl(C₁-C₄)alkyl, and unsubstituted aryloxy(C₁-C₄)alkyl. Typically, anaryl or heteroaryl group will have from zero to three substituents, withthose groups having two or fewer substituents being exemplary in thepresent invention. In one embodiment of the invention, an aryl orheteroaryl group will be unsubstituted or monosubstituted. In anotherembodiment, an aryl or heteroaryl group will be unsubstituted.

Two of the substituents on adjacent atoms of an aryl or heteroaryl ringin an aryl or heteroaryl group as described herein may optionally bereplaced with a substituent of the formula -T-C(O)—(CH₂)_(q)—U—, whereinT and U are independently —NH—, —O—, —CH₂— or a single bond, and q is aninteger of from 0 to 2. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -J-(CH₂)_(r)—K—, wherein J and K areindependently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR^(f)′—, ora single bond, and r is an integer of from 1 to 3. One of the singlebonds of the new ring so formed may optionally be replaced with a doublebond. Alternatively, two of the substituents on adjacent atoms of thearyl or heteroaryl ring may optionally be replaced with a substituent ofthe formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR^(f)′—, —S—, —S(O)—, —S(O)₂—,or —S(O)₂NR^(a)′—. The substituent R^(f)′ in —NR^(f)′— and—S(O)₂NR^(f)′— is selected from hydrogen or unsubstituted (C₁-C₆)alkyl.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

As used herein the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder to be prevented, reduced, or treated as described herein. Thephrase “therapeutically effective amount” as it relates to the GSNORinhibitors of the present invention shall mean the GSNOR inhibitordosage that provides the specific pharmacological response for which theGSNOR inhibitor is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a GSNOR inhibitor that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

The term “biological sample” includes, but is not limited to, samples ofblood (e.g., serum, plasma, or whole blood), urine, saliva, sweat,breast milk, vaginal secretions, semen, hair follicles, skin, teeth,bones, nails, or other secretions, body fluids, tissues, or cells. Inaccordance with the invention, the levels of the GSNOR in the biologicalsample can be determined by the methods described in U.S. PatentApplication Publication No. 2005/0014697.

D. Pharmaceutical Compositions

The invention encompasses pharmaceutical compositions comprising atleast one compound of the invention described herein and at least onepharmaceutically acceptable carrier. Suitable carriers are described in“Remington: The Science and Practice, Twentieth Edition,” published byLippincott Williams & Wilkins, which is incorporated herein byreference. Pharmaceutical compositions according to the invention mayalso comprise one or more non-inventive compound active agents.

The pharmaceutical compositions of the invention can comprise novelcompounds described herein, the pharmaceutical compositions can compriseknown compounds which previously were not known to have GSNOR inhibitoractivity, or a combination thereof.

The compounds of the invention can be utilized in any pharmaceuticallyacceptable dosage form, including, but not limited to injectable dosageforms, liquid dispersions, gels, aerosols, ointments, creams,lyophilized formulations, dry powders, tablets, capsules, controlledrelease formulations, fast melt formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, mixed immediate release and controlled releaseformulations, etc. Specifically, the compounds of the inventiondescribed herein can be formulated: (a) for administration selected fromthe group consisting of oral, pulmonary, intravenous, intra-arterial,intrathecal, intra-articular, rectal, ophthalmic, colonic, parenteral,intracisternal, intravaginal, intraperitoneal, local, buccal, nasal, andtopical administration; (b) into a dosage form selected from the groupconsisting of liquid dispersions, gels, aerosols, ointments, creams,tablets, sachets, and capsules; (c) into a dosage form selected from thegroup consisting of lyophilized formulations, dry powders, fast meltformulations, controlled release formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, and mixed immediate release and controlled releaseformulations; or (d) any combination thereof.

For respiratory infections, an inhalation formulation can be used toachieve high local concentrations. Formulations suitable for inhalationinclude dry power or aerosolized or vaporized solutions, dispersions, orsuspensions capable of being dispensed by an inhaler or nebulizer intothe endobronchial or nasal cavity of infected patients to treat upperand lower respiratory bacterial infections.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,or other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citrates,or phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes, or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may comprisesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The pharmaceutical composition should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating at least one compound of the invention into a sterilevehicle that contains a basic dispersion medium and any other requiredingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, both of which yield a powder of acompound of the invention plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the compound of the invention can be incorporated withexcipients and used in the form of tablets, troches, or capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash, wherein the compound in the fluid carrier is applied orallyand swished and expectorated or swallowed. Pharmaceutically compatiblebinding agents, and/or adjuvant materials can be included as part of thecomposition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In one embodiment, the compounds of the invention are prepared withcarriers that will protect against rapid elimination from the body. Forexample, a controlled release formulation can be used, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of the compounds of the invention may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also include suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of thecompound of the invention calculated to produce the desired therapeuticeffect in association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the invention are dictated byand directly dependent on the unique characteristics of the compound ofthe invention and the particular therapeutic effect to be achieved, andthe limitations inherent in the art of compounding such an active agentfor the treatment of individuals.

Pharmaceutical compositions according to the invention comprising atleast one compound of the invention can comprise one or morepharmaceutical excipients. Examples of such excipients include, but arenot limited to binding agents, filling agents, lubricating agents,suspending agents, sweeteners, flavoring agents, preservatives, buffers,wetting agents, disintegrants, effervescent agents, and otherexcipients. Such excipients are known in the art. Exemplary excipientsinclude: (1) binding agents which include various celluloses andcross-linked polyvinylpyrrolidone, microcrystalline cellulose, such asAvicel® PH101 and Avicel® PH102, silicified microcrystalline cellulose(ProSolv SMCC™), gum tragacanth and gelatin; (2) filling agents such asvarious starches, lactose, lactose monohydrate, and lactose anhydrous;(3) disintegrating agents such as alginic acid, Primogel, corn starch,lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch,and modified starches, croscarmellose sodium, cross-povidone, sodiumstarch glycolate, and mixtures thereof; (4) lubricants, including agentsthat act on the flowability of a powder to be compressed, includemagnesium stearate, colloidal silicon dioxide, such as Aerosil® 200,talc, stearic acid, calcium stearate, and silica gel; (5) glidants suchas colloidal silicon dioxide; (6) preservatives, such as potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid such as butylparaben, alcohols such asethyl or benzyl alcohol, phenolic compounds such as phenol, orquaternary compounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as Avicel® PH101 and Avicel® PH102;lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose®DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, Magnasweet® (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

E. Kits Comprising the Compositions of the Invention

The present invention also encompasses kits comprising the compositionsof the invention. Such kits can comprise, for example, (1) at least onecompound of the invention; and (2) at least one pharmaceuticallyacceptable carrier, such as a solvent or solution. Additional kitcomponents can optionally include, for example: (1) any of thepharmaceutically acceptable excipients identified herein, such asstabilizers, buffers, etc., (2) at least one container, vial, or similarapparatus for holding and/or mixing the kit components; and (3) deliveryapparatus, such as an inhaler, nebulizer, syringe, etc.

F. Methods of Preparing Compounds of the Invention

The compounds of the invention can readily be synthesized using knownsynthetic methodologies or via a modification of known syntheticmethodologies. As would be readily recognized by a skilled artisan, themethodologies described below allow the synthesis of thiophenes having avariety of substituents. Exemplary synthetic methods are described inthe examples below.

If needed, further purification and separation of enantiomers anddiastereomers can be achieved by routine procedures known in the art.Thus, for example, the separation of enantiomers of a compound can beachieved by the use of chiral HPLC and related chromatographictechniques. Diastereomers can be similarly separated. In some instances,however, diastereomers can simply be separated physically, such as, forexample, by controlled precipitation or crystallization.

The process of the invention, when carried out as prescribed herein, canbe conveniently performed at temperatures that are routinely accessiblein the art. In one embodiment, the process is performed at a temperaturein the range of about 25° C. to about 110° C. In another embodiment, thetemperature is in the range of about 40° C. to about 100° C. In yetanother embodiment, the temperature is in the range of about 50° C. toabout 95° C.

Synthetic steps that require a base are carried out using any convenientorganic or inorganic base. Typically, the base is not nucleophilic.Thus, in one embodiment, the base is selected from carbonates,phosphates, hydroxides, alkoxides, salts of disilazanes, and tertiaryamines.

The process of the invention, when performed as described herein, can besubstantially complete after several minutes to after several hoursdepending upon the nature and quantity of reactants and reactiontemperature. The determination of when the reaction is substantiallycomplete can be conveniently evaluated by ordinary techniques known inthe art such as, for example, HPLC, LCMS, TLC, and ¹H NMR.

G. Methods of Treatment

The invention encompasses methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through use ofone or more of the disclosed compounds. The methods compriseadministering a therapeutically effective amount of a compound of theinvention to a patient in need. The compositions of the invention canalso be used for prophylactic therapy.

The compound of the invention used in the methods of treatment accordingto the invention can be: (1) a novel compound described herein, or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, or a metabolite thereof; (2) a compound which was knownprior to the present invention, but wherein it was not known that thecompound is a GSNOR inhibitor, or a pharmaceutically acceptable saltthereof, a prodrug thereof, or a metabolite thereof; or (3) a compoundwhich was known prior to the present invention, and wherein it was knownthat the compound is a GSNOR inhibitor, but wherein it was not knownthat the compound is useful for the methods of treatment describedherein, or a pharmaceutically acceptable salt thereof, a prodrugthereof, or a metabolite thereof.

The patient can be any animal, domestic, livestock, or wild, including,but not limited to cats, dogs, horses, pigs, and cattle, and preferablyhuman patients. As used herein, the terms patient and subject may beused interchangeably.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition, ordisorder. More specifically, “treating” includes reversing, attenuating,alleviating, minimizing, suppressing, or halting at least onedeleterious symptom or effect of a disease (disorder) state, diseaseprogression, disease causative agent (e.g., bacteria or viruses), orother abnormal condition. Treatment is continued as long as symptomsand/or pathology ameliorate.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg/kg to 10 g/kg and often ranges from 10 μg/kg to 1 g/kgor 10 μg/kg to 100 mg/kg body weight of the subject being treated, perday.

H. GSNOR Uses

In subjects with deleteriously high levels of GSNOR or GSNOR activity,modulation may be achieved, for example, by administering one or more ofthe disclosed compounds that disrupts or down-regulates GSNOR function,or decreases GSNOR levels. These compounds may be administered withother GSNOR inhibitor agents, such as anti-GSNOR antibodies or antibodyfragments, GSNOR antisense, iRNA, or small molecules, or otherinhibitors, alone or in combination with other agents as described indetail herein.

The present invention provides a method of treating a subject afflictedwith a disorder ameliorated by NO donor therapy. Such a method comprisesadministering to a subject a therapeutically effective amount of a GSNORinhibitor.

The disorders can include pulmonary disorders associated with hypoxemiaand/or smooth muscle constriction in the lungs and airways and/or lunginfection and/or lung inflammation and/or lung injury (e.g., pulmonaryhypertension, ARDS, asthma, pneumonia, pulmonary fibrosis/interstitiallung diseases, cystic fibrosis, COPD); cardiovascular disease and heartdisease (e.g., hypertension, ischemic coronary syndromes,atherosclerosis, heart failure, glaucoma); diseases characterized byangiogenesis (e.g., coronary artery disease); disorders where there isrisk of thrombosis occurring; disorders where there is risk ofrestenosis occurring; inflammatory diseases (e.g., AIDS relateddementia, inflammatory bowel disease (IBD), Crohn's disease, colitis,and psoriasis); functional bowel disorders (e.g., irritable bowelsyndrome (IBS)); diseases where there is risk of apoptosis occurring(e.g., heart failure, atherosclerosis, degenerative neurologicdisorders, arthritis, and liver injury (ischemic or alcoholic));impotence; sleep apnea; diabetic wound healing; cutaneous infections;treatment of psoriasis; obesity caused by eating in response to cravingfor food; stroke; reperfusion injury (e.g., traumatic muscle injury inheart or lung or crush injury); and disorders where preconditioning ofheart or brain for NO protection against subsequent ischemic events isbeneficial, central nervous system (CNS) disorders (e.g., anxiety,depression, psychosis, and schizophrenia); and infections caused bybacteria (e.g., tuberculosis, C. difficile infections, among others).

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, or a prodrug or metabolitethereof, can be administered in combination with an NO donor. An NOdonor donates nitric oxide or a related redox species and more generallyprovides nitric oxide bioactivity, that is activity which is identifiedwith nitric oxide, e.g., vasorelaxation or stimulation or inhibition ofa receptor protein, e.g., ras protein, adrenergic receptor, NFκB. NOdonors including S-nitroso, O-nitroso, C-nitroso, and N-nitrosocompounds and nitro derivatives thereof and metal NO complexes, but notexcluding other NO bioactivity generating compounds, useful herein aredescribed in “Methods in Nitric Oxide Research,” Feelisch et al. eds.,pages 71-115 (J. S., John Wiley & Sons, New York, 1996), which isincorporated herein by reference. NO donors which are C-nitrosocompounds where nitroso is attached to a tertiary carbon which areuseful herein include those described in U.S. Pat. No. 6,359,182 and inWO 02/34705. Examples of S-nitroso compounds, including S-nitrosothiolsuseful herein, include, for example, S-nitrosoglutathione,S-nitroso-N-acetylpenicillamine, S-nitroso-cysteine and ethyl esterthereof, S-nitroso cysteinyl glycine,S-nitroso-gamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin. Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, isosorbide, nitroglycerin, SIN 1which is molsidomine, furoxamines, N-hydroxy (N-nitrosamine), andperfluorocarbons that have been saturated with NO or a hydrophobic NOdonor.

The combination of a GSNOR inhibitor with R(+) enantiomer of amlodipine,a known NO releaser (Zhang at al., J. Cardiovasc. Pharm. 39: 208-214(2002)) is also an embodiment of the present invention.

The present invention also provides a method of treating a subjectafflicted with pathologically proliferating cells where the methodcomprises administering to said subject a therapeutically effectiveamount of an inhibitor of GSNOR. The inhibitors of GSNOR are thecompounds as defined above, or a pharmaceutically acceptable saltthereof, or a prodrug or metabolite thereof, in combination with apharmaceutically acceptable carrier. Treatment is continued as long assymptoms and/or pathology ameliorate.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating microbes. The microbes involved can bethose where GSNOR is expressed to protect the microbe from nitrosativestress or where a host cell infected with the microbe expresses theenzyme, thereby protecting the microbe from nitrosative stress. The term“pathologically proliferating microbes” is used herein to meanpathologic microorganisms including, but not limited to, pathologicbacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa,pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata.More detail on the applicable microbes is set forth at columns 11 and 12of U.S. Pat. No. 6,057,367. The term “host cells infected withpathologic microbes” includes not only mammalian cells infected withpathologic viruses but also mammalian cells containing intracellularbacteria or protozoa, e.g., macrophages containing Mycobacteriumtuberculosis, Mycobacterium leper (leprosy), or Salmonella typhi(typhoid fever).

In another embodiment, the pathologically proliferating cells can bepathologic helminths. The term “pathologic helminths” is used herein torefer to pathologic nematodes, pathologic trematodes and pathologiccestodes. More detail on the applicable helminths is set forth at column12 of U.S. Pat. No. 6,057,367.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating mammalian cells. The term “pathologicallyproliferating mammalian cells” as used herein means cells of the mammalthat grow in size or number in said mammal so as to cause a deleteriouseffect in the mammal or its organs. The term includes, for example, thepathologically proliferating or enlarging cells causing restenosis, thepathologically proliferating or enlarging cells causing benign prostatic hypertrophy, the pathologically proliferating cells causingmyocardial hypertrophy, and proliferating cells at inflammatory sitessuch as synovial cells in arthritis or cells associated with a cellproliferation disorder.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the term “psoriatic condition” refers to disordersinvolving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration. The cell proliferative disordercan be a precancerous condition or cancer. The cancer can be primarycancer or metastatic cancer, or both.

As used herein, the term “cancer” includes solid tumors, such as lung,breast, colon, ovarian, pancreas, prostate, adenocarcinoma, squamouscarcinoma, sarcoma, malignant glioma, leiomyosarcoma, hepatoma, head andneck cancer, malignant melanoma, non-melanoma skin cancers, as well ashematologic tumors and/or malignancies, such as leukemia, childhoodleukemia and lymphomas, multiple myeloma, Hodgkin's disease, lymphomasof lymphocytic and cutaneous origin, acute and chronic leukemia such asacute lymphoblastic, acute myelocytic, or chronic myelocytic leukemia,plasma cell neoplasm, lymphoid neoplasm, and cancers associated withAIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses, andthe like. In one embodiment, proliferative diseases include dysplasiasand disorders of the like.

In one embodiment, treating cancer comprises a reduction in tumor size,decrease in tumor number, a delay of tumor growth, decrease in metastaiclesions in other tissues or organs distant from the primary tumor site,an improvement in the survival of patients, or an improvement in thequality of patient life, or at least two of the above.

In another embodiment, treating a cell proliferative disorder comprisesa reduction in the rate of cellular proliferation, reduction in theproportion of proliferating cells, a decrease in size of an area or zoneof cellular proliferation, or a decrease in the number or proportion ofcells having an abnormal appearance or morphology, or at least two ofthe above.

In yet another embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ormetabolite thereof can be administered in combination with a secondchemotherapeutic agent. In a further embodiment, the secondchemotherapeutic agent is selected from the group consisting oftamoxifen, raloxifene, anastrozole, exemestane, letrozole, cisplatin,carboplatin, paclitaxel, cyclophosphamide, lovastatin, minosine,gemcitabine, araC, 5-fluorouracil, methotrexate, docetaxel, goserelin,vincristin, vinblastin, nocodazole, teniposide, etoposide, epothilone,navelbine, camptothecin, daunonibicin, dactinomycin, mitoxantrone,amsacrine, doxorubicin, epirubicin, idarubicin imatanib, gefitinib,erlotinib, sorafenib, sunitinib malate, trastuzumab, rituximab,cetuximab, and bevacizumab.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a prodrug thereof, ametabolite thereof can be administered in combination with an agent thatimposes nitrosative or oxidative stress. Agents for selectively imposingnitrosative stress to inhibit proliferation of pathologicallyproliferating cells in combination therapy with GSNOR inhibitors hereinand dosages and routes of administration therefor include thosedisclosed in U.S. Pat. No. 6,057,367, which is incorporated herein.Supplemental agents for imposing oxidative stress (i.e., agents thatincrease GSSG (oxidized glutathione) over GSH (glutathione) ratio orNAD(P) over NAD(P)H ratio or increase thiobarbituric acid derivatives)in combination therapy with GSNOR inhibitors herein include, forexample, L-buthionine-5-sulfoximine (BSO), glutathione reductaseinhibitors (e.g., BCNU), inhibitors or uncouplers of mitochondrialrespiration, and drugs that increase reactive oxygen species (ROS),e.g., adriamycin, in standard dosages with standard routes ofadministration.

GSNOR inhibitors may also be co-administered with a phosphodiesteraseinhibitor (e.g., rolipram, cilomilast, roflumilast, Viagra® (sildenifilcitrate), Clalis® (tadalafil), Levitra® (vardenifil), etc.), aβ-agonist, a steroid, or a leukotriene antagonist (LTD-4). Those skilledin the art can readily determine the appropriate therapeuticallyeffective amount depending on the disorder to be ameliorated.

GSNOR inhibitors may be used as a means to improve β-adrenergicsignaling. In particular, inhibitors of GSNOR alone or in combinationwith β-agonists could be used to treat or protect against heart failure,or other vascular disorders such as hypertension and asthma. GSNORinhibitors can also be used to modulate G protein coupled receptors(GPCRs) by potentiating Gs G-protein, leading to smooth musclerelaxation (e.g., airway and blood vessels), and by attenuating GqG-protein, and thereby preventing smooth muscle contraction (e.g., inairway and blood vessels).

The therapeutically effective amount for the treatment of a subjectafflicted with a disorder ameliorated by NO donor therapy is the GSNORinhibiting amount in vivo that causes amelioration of the disorder beingtreated or protects against a risk associated with the disorder. Forexample, for asthma, a therapeutically effective amount is abronchodilating effective amount; for cystic fibrosis, a therapeuticallyeffective amount is an airway obstruction ameliorating effective amount;for ARDS, a therapeutically effective amount is a hypoxemia amelioratingeffective amount; for heart disease, a therapeutically effective amountis an angina relieving or angiogenesis inducing effective amount; forhypertension, a therapeutically effective amount is a blood pressurereducing effective amount; for ischemic coronary disorders, atherapeutic amount is a blood flow increasing effective amount; foratherosclerosis, a therapeutically effective amount is an endothelialdysfunction reversing effective amount; for glaucoma, a therapeuticamount is an intraocular pressure reducing effective amount; fordiseases characterized by angiogenesis, a therapeutically effectiveamount is an angiogenesis inhibiting effective amount; for disorderswhere there is risk of thrombosis occurring, a therapeutically effectiveamount is a thrombosis preventing effective amount; for disorders wherethere is risk of restenosis occurring, a therapeutically effectiveamount is a restenosis inhibiting effective amount; for chronicinflammatory diseases, a therapeutically effective amount is aninflammation reducing effective amount; for disorders where there isrisk of apoptosis occurring, a therapeutically effective amount is anapoptosis preventing effective amount; for impotence, a therapeuticallyeffective amount is an erection attaining or sustaining effectiveamount; for obesity, a therapeutically effective amount is a satietycausing effective amount; for stroke, a therapeutically effective amountis a blood flow increasing or a TIA protecting effective amount; forreperfusion injury, a therapeutically effective amount is a functionincreasing effective amount; and for preconditioning of heart and brain,a therapeutically effective amount is a cell protective effectiveamount, e.g., as measured by troponin or CPK.

The therapeutically effective amount for the treatment of a subjectafflicted with pathologically proliferating cells means a GSNORinhibiting amount in vivo which is an antiproliferative effectiveamount. Such antiproliferative effective amount as used herein means anamount causing reduction in rate of proliferation of at least about 20%,at least about 10%, at least about 5%, or at least about 1%.

I. Uses in an Apparatus

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug, or a stereoisomer, or metabolite thereof,can be applied to various apparatus in circumstances when the presenceof such compounds would be beneficial. Such apparatus can be any deviceor container, for example, implantable devices in which a compound ofthe invention can be used to coat a surgical mesh or cardiovascularstent prior to implantation in a patient. The compounds of the inventioncan also be applied to various apparatus for in vitro assay purposes orfor culturing cells.

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a prodrug, or a stereoisomer, or metabolite thereof canalso be used as an agent for the development, isolation or purificationof binding partners to compounds of the invention, such as antibodies,natural ligands, and the like. Those skilled in the art can readilydetermine related uses for the compounds of the present invention.

EXAMPLES

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

Examples 1-14 list representative novel thiophene analogs of Formula Iuseful as GSNOR inhibitors of the invention. Exemplary schemes belowillustrate some general methods of making the thiophene analogs of theinvention. Synthetic methods that can be used to prepare each compoundare described in Examples 1-14. Supporting mass spectrometry data and/orproton NMR data for each compound is also included in Examples 1-14.Synthetic details for corresponding Intermediates are detailed inExample 15.

See Example 1 below for a representative detailed description of SchemeI.

See Example 4 for a representative detailed description of Scheme II.

See Example 14 for a representative detailed description of Scheme III.

Example 13-(3-(4-carbamoyl-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)propanoicacid

Step 1 of General Scheme I: Synthesis of3-bromo-4-(4-methoxyphenyl)thiophene (2, X₁=4-methoxyphenyl)

To a mixture of 3,4-dibromothiophene (Scheme I, 1) (5 g, 20.7 mmol),4-methoxyphenylboronic acid (Scheme I, 2, X₁=4-methoxyphenyl) (3.14 g,20.7 mmol), sodium carbonate (4.38 g, 41.3 mmol), toluene (100 mL), in amixture of ethanol (60 mL) and water (40 mL) was added Pd(PPh₃)₄ (2.02g, 1.75 mmol) under nitrogen. The mixture was stirred at 75° C. for 13h, poured into water (60 mL), and extracted with ethyl acetate (60mL×3). The organic extracts were dried over anhydrous magnesium sulfate(10 g), evaporated, and purified by column chromatography on silica gel(petroleum ether/ethyl acetate=150:1) to give3-bromo-4-(4-methoxyphenyl)thiophene (Scheme I, 2, X₁=4-methoxyphenyl)(1.7 g, yield 30.6%). ¹H NMR (DMSO-d₆ 400 MHz): δ 7.84 (d, J=3.2 Hz,1H), 7.64 (d, J=3.2 Hz, 1H), 7.42 (d, J=8.8 Hz, 2H), 6.97 (d, J=8.8 Hz,2H), 3.78 (s, 3H).

Step 2 of General Scheme I: Synthesis of4-(4-(4-methoxyphenyl)thiophen-3-yl)-3-methylbenzonitrile (3,X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl)

To a mixture of 3-bromo-4-(4-methoxyphenyl)thiophene (Scheme I, 2,X₁=4-methoxyphenyl) (1.7 g, 6.316 mmol), 4-cyano-2-methylphenylboronicacid (Scheme I, 2A, X₂=4-cyano-2-methylphenyl) (1.535 g, 6.316 mmol),sodium carbonate (1.34 g, 12.63 mmol), toluene (60 mL) in a mixture ofethanol (36 mL) and water (24 mL) under nitrogen was added Pd(PPh₃)₄(0.7 g, 0.606 mmol). The mixture was stirred at 80° C. for 5 h, pouredinto water (50 mL), and extracted with ethyl acetate (50 mL×3). Theorganic extracts were dried over anhydrous magnesium sulfate (6 g),evaporated, and purified by column chromatography on silica gel(petroleum ether/ethyl acetate=50:1) to give4-(4-(4-methoxyphenyl)thiophen-3-yl)-3-methylbenzonitrile (Scheme I, 3,X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (1.34 g, yield 69.4%). ¹HNMR (DMSO-d₆ 400 MHz): δ 7.68-7.65 (m, 3H), 7.58 (d, J=3.2 Hz, 1H), 7.33(d, J=8.0 Hz, 1H), 6.98 (d, J=8.8 Hz, 2H), 6.80 (d, J=8.8 Hz, 2H), 3.70(s, 3H), 1.79 (s, 3H).

Step 3 of General Scheme I: Synthesis of4-(2-formyl-4-(4-methoxyphenyl)thiophen-3-yl)-3-methylbenzonitrile (4B,X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl)

A solution of freshly prepared LDA (0.8 mL, 1.64 mmol) in anhydroustetrahydrofuran (7 mL) was added slowly a solution of4-(4-(4-methoxyphenyl)thiophen-3-yl)-3-methylbenzonitrile (Scheme I, 3,X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (500 mg, 1.637 mmol) inanhydrous tetrahydrofuran (6 mL) under nitrogen at −78° C. After beingstirred for 15 min., a solution of dimethylformamide (0.14 mL, 1.804mmol) in anhydrous tetrahydrofuran (3 mL) was dropwise added. Theresulting mixture was stirred for 1 h at −78° C., and at roomtemperature for 3 h. The reaction was quenched with water (50 mL), andextracted with ethyl acetate (40 mL×3). The combined organic extractswere washed with water (30 mL), brine (50 mL), dried over anhydroussodium sulfate, evaporated, and purified by column chromatography(silica gel, petroleum ether/ethyl acetate=20:1) to afford4-(2-formyl-4-(4-methoxyphenyl)thiophen-3-yl)-3-methylbenzonitrile(Scheme I, 4A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (0.16 g,yield 29.3%) as a white solid (confirmed by NOE). ¹H NMR (DMSO-d₆ 400MHz): δ 9.46 (s, 1H), 8.31 (d, J=1.2 Hz, 1H), 7.83 (s, 2H), 7.67 (d,J=8.8 Hz, 1H), 7.07 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 3.77 (s,3H), 1.94 (s, 3H).

Step 4 of General Scheme I: Synthesis of (E)-ethyl3-(3-(4-cyano-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)acrylate(5A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl)

The mixture of compound4-(2-formyl-4-(4-methoxyphenyl)thiophen-3-yl)-3-methylbenzonitrile(Scheme I, 4A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (0.16 g,0.48 mmol) and (carbethoxymethylene)triphenylphosphorane (Ph₃P═CHCOOEt)(0.17 g, 0.48 mmol) in toluene (15 mL) was heated to 100° C. for 14 h.After being cooled down to room temperature, the reaction mixture wasconcentrated and purified by silica gel column chromatography (petroleumether/ethyl acetate=20:1) to afford (E)-ethyl3-(3-(4-cyano-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)acrylate(Scheme I, 5A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (0.27 g,yield 100%) as a white solid.

Step 5 of General Scheme I: Synthesis of ethyl3-(3-(4-cyano-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)propanoate(6A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl)

To a solution of (E)-ethyl3-(3-(4-cyano-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)acrylate(Scheme I, 5A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (0.27 g,0.669 mmol) in ethanol (40 mL) was added 10% Pd/C (0.25 g), and thereaction mixture was stirred at 20° C. under 20 Psi of hydrogen for 30h. The reaction mixture was filtrated, and concentrated to give ethyl3-(3-(4-cyano-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)propanoate(Scheme I, 6A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (0.2 g,yield 73.7%). ¹H NMR (DMSO-d₆ 300 MHz): δ 7.71 (m, 2H), 7.45-7.40 (m,2H), 6.92 (m, 2H), 6.75 (m, 2H), 4.03-3.96 (m, 2H), 3.66 (s, 3H),2.76-2.71 (m, 3H), 1.80 (s, 2H), 1.22 (s, 1H), 1.14-1.09 (m, 3H).

Step 6 of General Scheme I: Synthesis of3-(3-(4-carbamoyl-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)propanoicacid (7A, X₁=4-methoxyphenyl, X₂=4-carbamoyl-2-methylphenyl)

To a mixture of ethyl3-(3-(4-cyano-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)propanoate(Scheme I, 6A, X₁=4-methoxyphenyl, X₂=4-cyano-2-methylphenyl) (180 mg,0.44 mmol), sodium hydroxide (35.52 mg, 0.88 mmol) in a mixture of DMSO(10 mL) and water (3 mL) was added 30% hydrogen peroxide (0.3 mL). Thereaction mixture was stirred at 20° C. for 5 h, hydrogen chloride (1.2mL, 1 mol/L) was added until pH=5, the resulting mixture was extractedwith ethyl acetate (10 mL×4). The combined organic layer was washed withbrine (15 mL), dried over anhydrous sodium sulfate, evaporated, andpurified by preparative HPLC to give Example 1 (Scheme I, 7A,X₁=4-methoxyphenyl, X₂=4-carbamoyl-2-methylphenyl) (80 mg, yield 45.6%).¹H NMR (DMSO-d₆ 400 MHz): δ 12.20 (br s, 1H), 7.93 (s, 1H), 7.71-7.67(m, 2H), 7.40 (s, 1H), 7.931 (s, 1H), 7.23 (d, J=8.0 Hz, 1H), 6.94 (d,J=8.4 Hz, 2H), 6.71 (d, J=8.4 Hz, 2H), 3.64 (s, 3H), 2.72 (m, 2H), 2.40(m, 2H), 1.81 (s, 3H); MS (ESI): m/z 396.1 [M+H]⁺.

Example 23-(3-(4-carbamoylphenyl)-4-(4-chloro-2-methoxyphenyl)thiophen-2-yl)propanoicacid

Prepared following General Scheme I. The starting material 1A in Step 1was 4-cyano-phenylboronic acid, and the crude was purified by columnchromatography (PE: EtOAc=20:1) to give4-(4-bromothiophen-3-yl)benzonitrile (8 g, yield 37%). The startingmaterial 2A in Step 2 was 4-chloro-2-methoxyphenylboronic acid, thereaction was heated at 100° C. overnight, and crude was purified bycolumn chromatography (PE:EtOAc=20:1) to give4-(4-(4-chloro-2-methoxyphenyl)thiophen-3-yl)benzonitrile (0.9 g, yield73%). Steps 3, 4, 5 and 6 were performed as described in Scheme 1. Bothisomers formed in Step 3 were carried through the entire synthesis andwere separated after the last step by HPLC. ¹H NMR (CD₃OD 400 MHz): δ7.78 (d, J=8.4 Hz, 2H), 7.20-7.14 (m, 4H), 6.90 (d, J=8.0 Hz, 1H), 6.78(s, 1H), 3.11 (t, J=7.6 Hz, 2H), 2.29 (t, J=7.6 Hz, 2H); MS (ESI): m/z438.0 [M+23]⁺.

Example 33-(3-(4-chloro-2-methoxyphenyl)-4-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid

Prepared following General Scheme I. The starting material 1A in Step 1was 4-hydroxyphenylboronic acid and purification by columnchromatography (PE:EtOAc=20:1) gave 4-(4-bromothiophen-3-yl)phenol (2 g,38% yield). The starting material 2A in Step 2 was4-chloro-2-methoxyphenylboronic acid and purification by columnchromatography (PE: EtOAc=20:1) gave4-(4-(4-chloro-2-methoxyphenyl)thiophen-3-yl)phenol (0.8 g, yield 65%).After Step 2, the hydroxyl group was protected according to thefollowing description.

Synthesis of 3-(4-chloro-2-methoxyphenyl)-4-(4-(methoxymethoxy)phenyl)thiophene

To a solution of 4-(4-(4-chloro-2-methoxyphenyl)thiophen-3-yl)phenol(700 mg, 2.1 mmol) in anhydrous THF (5 mL) was added NaH (90 mg, 2.3mmol) at 0° C. After the mixture was stirred at the temperature for 0.5h, chloro (methoxy) methane (184 mg, 2.3 mmol) was added. The mixturewas stirred at 0° C. for 1 hour, and at room temperature for 2 hours.When TLC indicated the starting material was consumed, the mixture wasquenched with water, and extracted with EtOAc. The organic layer wasdried over Na₂SO₄ and concentrated to give the crude product, which waspurified by column to give3-(4-chloro-2-methoxyphenyl)-4-(4-(methoxymethoxy)phenyl)thiophene (720mg, 90%) which was taken forward to Step 3.

Followed Step 3 described in Scheme I, to give3-(4-chloro-2-methoxyphenyl)-4-(4-(methoxymethoxy)phenyl)thiophene-2-carbaldehyde(290 mg, yield 37%). Followed Step 4 described in Scheme 1, where crudewas purified by column chromatography (PE:EtOAc=15:1) to give (E)-ethyl3-(3-(4-chloro-2-methoxyphenyl)-4-(4-(methoxymethoxy)phenyl)thiophen-2-yl)acrylate(280 mg, yield 82%). Step 5 of Scheme 1 gave ethyl3-(3-(4-chloro-2-methoxyphenyl)-4-(4-(methoxymethoxy)phenyl)thiophen-2-yl)propanoate(220 mg, yield 78%). Step 6 of Scheme 1 gave crude3-(3-(4-chloro-2-methoxyphenyl)-4-(4-(methoxymethoxy)phenyl)thiophen-2-yl)propanoicacid (190 mg, yield 92%). The final step was a deprotection of thealcohol and is described here.

Synthesis of3-(3-(4-chloro-2-methoxyphenyl)-4-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid

To a solution of3-(3-(4-chloro-2-methoxyphenyl)-4-(4-(methoxymethoxy)phenyl)thiophen-2-yl)propanoicacid (190 mg, 0.44 mmol) in THF (5 mL) was added HCl (1 mL, 12 M) andthe mixture was stirred at room temperature for 2 hours. The mixture wasdiluted with water, extracted with EtOAc, and concentrated to give thecrude compound, which was purified by preparative HPLC (Column: YMCODS-AQ 150×30 cm, 5 μm; Retention Time: 15 min; Mobile phase: from 38%MeCN in water (0.05% TFA) to 68% MeCN in water (0.05% TFA); Wavelength:220 nm, 254 nm; sample dissolved in DMSO) to give3-(3-(4-chloro-2-methoxyphenyl)-4-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid (47.5 mg, yield 28%). ¹H NMR (CD₃OD 400 MHz): δ 7.11 (s, 1H), 6.99(d, J=1.6 Hz, 1H), 6.97-6.92 (m, 2H), 6.89 (d, J=8.4 Hz, 2H), 6.58 (d,J=8.8 Hz, 2H), 3.58 (s, 3H), 2.90 (t, J=8.0 Hz, 2H), 2.50 (t, J=8.0 Hz,2H); MS (ESI): m/z [M+1]⁺=389.1.

Example 43-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid

Step 1 of General Scheme II: Synthesis of4-bromo-3-(4-hydroxyphenyl)thiophene-2-carbaldehyde

A mixture of 3,4-dibromothiophene-2-carbaldehyde (see Intermediate 1(see Example 15 for Intermediate synthesis)) (2.0 g, 7.5 mmol),4-hydroxyphenylboronic acid (Scheme II, 8A, X₁=4-hydroxyphenyl) (1.0 g,7.5 mmol), Na₂CO₃ (1.6 g, 15 mmol), and Pd(PPh₃)₄ (431 mg, 0.37 mmol) intoluene/EtOH/H₂O (30 mL, 5:3:2) was heated to 80° C. and stirredovernight under nitrogen. When TLC indicated that the starting materialwas consumed, the mixture was diluted with water, neutralized to pH=4-5with HCl (1M), extracted with EtOAc, concentrated, and purified bycolumn chromatography (PE:EtOAc=20:1) to give4-bromo-3-(4-hydroxyphenyl)thiophene-2-carbaldehyde (Scheme II, 8,X₁=4-hydroxyphenyl) (350 mg, yield 17%) as a white solid. ¹H NMR(DMSO-d₆ 400 MHz): δ9.90 (s, 1H), 9.53 (s, 1H), 8.33 (s, 1H), 7.33 (d,J=8.4 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H).

Step 2 of General Scheme II: Synthesis of4-bromo-3-(4-hydroxyphenyl)thiophene-2-carbaldehyde

A mixture of 4-bromo-3-(4-hydroxyphenyl)thiophene-2-carbaldehyde (SchemeII, 8, X₁=4-hydroxyphenyl) (300 mg, 1.1 mmol),4-chloro-2-methoxyphenylboronic acid (Scheme II, 9A,X₂=4-chloro-2-methoxyphenyl) (237 mg, 1.3 mmol), Na₂CO₃ (225 mg, 2.2mmol), and Pd(PPh₃)₄ (61 mg, 0.05 mmol) in toluene/EtOH/H₂O (10 mL,5:3:2) was heated at 100° C., and stirred overnight under nitrogen. WhenTLC indicated that the starting material was consumed, the mixture wasdiluted with water, neutralized to pH=4-5 with HCl (1M), extracted withEtOAc, concentrated, and purified by column chromatography(PE:EtOAc=20:1) to give4-bromo-3-(4-hydroxyphenyl)thiophene-2-carbaldehyde (Scheme II, 9,X₁=4-hydroxyphenyl, X₂=4-chloro-2-methoxyphenyl) (290 mg, yield 79%) asa white solid. ¹H NMR (DMSO-d₆ 400 MHz): δ 9.54 (s, 1H), 9.50 (s, 1H),7.90 (s, 1H), 6.98 (d, J=8.0 Hz, 1H), 6.89 (m, 4H), 6.58 (d, J=8.4 Hz,2H), 3.29 (s, 3H).

Step 3 of General Scheme II: Synthesis of (E)-ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)acrylate

A mixture of 4-bromo-3-(4-hydroxyphenyl)thiophene-2-carbaldehyde (290mg, 0.84 mmol) and (carbethoxymethylene) triphenylphosphorane (322 mg,0.93 mmol) in toluene (10 mL) was stirred at 100° C. overnight. When TLCindicated the starting material was consumed, the mixture wasconcentrated and purified by column chromatography (PE:EtOAc=15:1) togive (E)-ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)acrylate(Scheme II, 10, X₁=4-hydroxyphenyl, X₂=4-chloro-2-methoxyphenyl) (280mg, yield 80%). ¹H NMR (DMSO-d₆ 400 MHz TMS): δ9.56 (s, 1H), 7.65 (s,1H), 7.51 (d, J=15.6 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 6.95 (d, J=8.0 Hz,2H), 6.81 (d, J=8.4 Hz, 2H), 6.71 (d, J=8.4 Hz, 2H), 6.26 (d, J=15.6 Hz,1H), 4.13 (q, J=7.2 Hz, 2H), 3.41 (s, 3H), 1.20 (t, J=7.2 Hz, 3H).

Step 4 of General Scheme II: Synthesis of ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoate

To a solution of (E)-ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)acrylate(280 mg, 0.61 mmol) in EtOH (5 mL) was added Pd/C (50 mg). After themixture was stirred at room temperature under H₂ atmosphere for 4 hours,the mixture was filtered and concentrated in vacuo to give ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoate(Scheme II, 11, X₁=4-hydroxyphenyl, X₂=4-chloro-2-methoxyphenyl) (220mg, yield 78%). ¹H NMR (DMSO-d₆ 400 MHz): δ 9.34 (s, 1H), 7.26 (s, 1H),7.01 (d, J=8.4 Hz, 1H), 6.90 (d, J=8.8 Hz, 2H), 6.79 (d, J=8.0 Hz, 2H),6.64 (d, J=8.4 Hz, 2H), 4.04 (q, J=7.2 Hz, 2H), 3.40 (s, 3H), 2.96 (t,J=7.6 Hz, 2H), 2.55 (t, J=7.6 Hz, 2H), 1.16 (t, J=7.2 Hz, 3H).

Step 5 of General Scheme II: Synthesis of3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid

To a solution of ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoate(220 mg, 0.53 mmol) in EtOH (5 mL) was added NaOH (42 mg, 1.06 mmol),and the mixture was stirred at room temperature for 2 hours. The mixturewas diluted with water, neutralized to pH=4-5, extracted with EtOAc, andconcentrated to give3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid (Scheme II, 12, X₁=4-hydroxyphenyl, X₂=4-chloro-2-methoxyphenyl)(127.42 mg, yield 62%). ¹H NMR (DMSO-d₆ 400 MHz): δ 9.36 (br, 1H), 7.25(s, 1H), 7.01 (d, J 8.0 Hz, 1H), 6.91-6.88 (m, 2H), 6.79 (d, J=8.4 Hz,2H), 6.64 (d, J=8.4 Hz, 2H), 3.40 (s, 3H), 2.92 (t, J=7.2 Hz, 2H), 2.47(t, J=7.2 Hz, 2H); MS (ESI): m/z [M+1]⁺=389.0.

Example 53-(4-(4-chloro-2-methoxyphenyl)-3-(4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoicacid

Prepared following General Scheme II. The starting material 8A in Step 1was 4-amino-2-methylphenylboronic acid, and the crude was purified bycolumn chromatography (PE: EtOAc=5:1) to give3-(4-aminophenyl)-4-bromothiophene-2-carbaldehyde (1.5 g, yield 47.8%).The starting material 9A in Step 2 was 4-chloro-2-methoxyphenylboronicacid, and crude was purified by column chromatography (PE:EtOAc=3:1) togive3-(4-aminophenyl)-4-(4-chloro-2-methoxyphenyl)thiophene-2-carbaldehyde(300 mg, yield, 49.3%). Step 3 of Scheme II was followed, with crudepurified by column chromatography (PE:EtOAc=3:1) to give (E)-ethyl3-(3-(4-aminophenyl)-4-(4-chloro-2-methoxyphenyl)thiophen-2-yl)acrylate(300 mg, yield 83%). The amine of this product was then mesylatedfollowing the procedure described here.

Synthesis of (E)-ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-(methylsulfonamido)phenyl)thiophen-2-yl)acrylate

To a mixture of (E)-ethyl3-(3-(4-aminophenyl)-4-(4-chloro-2-methoxyphenyl)thiophen-2-yl)acrylate(300 mg, 0.73 mmol) and TEA (75 mg, 0.73 mmol) in DCM (10 mL) was addedMsCl (108 mg, 0.95 mmol), and the mixture was stirred at roomtemperature overnight. The reaction solution was washed with brine,dried over sodium sulfate, and concentrated under reduced pressure togive (E)-ethyl 3-(4-(4-chloro-2-methoxyphenyl)-3-(4-(methylsulfonamido)phenyl)thiophen-2-yl)acrylate, which was used in the next step withoutfurther purification.

Following Step 4 of General Scheme II gave ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoate(200 mg, yield 80.0%). Following Step 5 of Scheme II, where the mixturewas acidified to pH=7, and purified by preparative HPLC gave 29 mg ofExample 5 (yield 15.3%). ¹H NMR (CD₃OD, 400 MHz): δ 7.15-7.17 (m, 3H),7.10 (d, J=8.0 Hz, 1H), 7.03 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.4 Hz, 1H),6.80 (s, 1H), 3.40 (s, 3H), 3.09 (t, J=7.6 Hz, 2H), 2.58 (t, J=7.6 Hz,2H); MS (ESI): m/z 488.1 [M+1]⁺.

Example 63-(4-(4-carbamoylphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoicacid

Followed the procedure described in General Scheme II, withmodification. Step 1 was followed where the starting material was4-(4-bromo-5-formylthiophen-3-yl)benzonitrile (Intermediate 3) insteadof 3,4-dibromothiophene-2-carbaldehyde (Intermediate I) and startingmaterial 8A was Intermediate 2(2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-imidazole).After purification by column chromatography (DCM:MeOH=30:1) the desired4-(5-formyl-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-3-yl)benzonitrile(500 mg, yield 60%) was obtained. Step 3 of Scheme II was then followedwith column conditions (PE:EtOAc=1:2) to afford (E)-ethyl3-(4-(4-cyanophenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)acrylate(600 mg, yield 100%). Step 4 of Scheme II gave 450 mg of product (yield75%) followed by Step 5 (with purification by prep-HPLC) gave Example 6(45.98 mg, 10% yield). ¹H NMR (CD₃OD 400 MHz): δ 7.65-7.63 (m, 3H), 7.53(s, 1H), 7.47 (d, J=8.0 Hz, 2H), 7.38-7.34 (m, 3H), 7.13 (d, J=8.0 Hz,2H), 3.04 (t, J=7.6 Hz, 2H), 2.56-2.52 (m, 5H); MS (ESI): m/z 432.0[M+1]⁺.

Example 73-(3-(4-carbamoyl-2-methylphenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoicacid

Prepared following General Scheme II. The starting material 8A in Step 1was 4-cyano-2-methylphenylboronic acid, and column conditions for Step 1was (PE: EtOAc=10:1) to give4-(4-bromo-2-formylthiophen-3-yl)-3-methylbenzonitrile (2 g, yield 88%).The starting material 9A in Step 2 was Intermediate 2(2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-imidazole),and crude was purified by column chromatography (PE:EtOAc=1:2) to give4-(2-formyl-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-3-yl)-3-methylbenzonitrile(0.55 g, yield 60%). Step 3 of scheme II was followed, with purificationby column chromatography (PE:EtOAc=1:1) to afford (E)-ethyl3-(3-(4-cyano-2-methylphenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)acrylate(600 mg, yield 92%). Following Step 4 of General Scheme II gave 400 mgof desired (66% yield). Following Step 5 of Scheme II, where crude waspurified by preparative HPLC, gave 26.4 mg of Example 7 (yield 9%). ¹HNMR (DMSO-d₆ 400 MHz): δ 7.99 (s, 1H), 7.87 (s, 1H), 7.79-7.73 (m, 4H),7.47 (d, J=8.4 Hz, 2H), 7.39 (s, 1H), 7.32-7.30 (m, 3H), 2.84-2.69 (m,2H), 2.50-2.42 (m, 5H), 1.89 (s, 3H); MS (ESI): m/z 446.1 [M+1]⁺.

Example 83-(3-(4-carbamoylphenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoicacid

Prepared following General Scheme II. The starting material 8A in Step 1was 4-cyanophenylboronic acid. The starting material 9A in Step 2 wasIntermediate 2(2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-imidazole),and crude was purified by column chromatography (PE:EtOAc=2:1) to give4-(2-formyl-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-3-yl)benzonitrile(300 mg, yield 47%). Step 3 of scheme II was followed, with purificationby column chromatography (PE:EtOAc=1:2) to afford (E)-ethyl3-(3-(4-cyanophenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)acrylate(250 mg, yield 70%). Following Step 4 of General Scheme II gave ethyl3-(3-(4-cyanophenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoate(200 mg, yield 80%) and Step 5 of Scheme II, where crude was purified bypreparative HPLC gave 166 mg of Example 8 (yield 85%). ¹H NMR (CD₃OD 300MHz): δ 7.88 (d, J=8.4 Hz, 2H), 7.64 (d, J=2.1 Hz, 1H), 7.58 (d, J=2.1Hz, 1H), 7.50 (s, 1H), 7.43-7.35 (m, 4H), 7.29 (d, J=8.4 Hz, 2H), 3.09(t, J=7.2 Hz, 2H), 2.61-2.55 (m, 5H); MS (ESI): m/z 432.0 [M+1]⁺.

Example 93-(4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-3-(2-methyl-4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoicacid

Followed the procedure described in General Scheme II, withmodification. Step 1 was followed where the starting material wasN-(4-(4-bromo-2-formylthiophen-3-yl)-3-methylphenyl)methanesulfonamide(Intermediate 4) instead of 3,4-dibromothiophene-2-carbaldehyde(Intermediate I) and starting material 8A was Intermediate 2(2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-imidazole).After purification by column chromatography (PE:EtOAc=1:2)N-(4-(2-formyl-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-3-yl)-3-methylphenyl)methanesulfonamide(200 mg, yield 30%) was obtained. Step 3 of Scheme II was then followedwith column conditions (PE:EtOAc=1:2) to afford (E)-ethyl3-(4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-3-(4-(methylsulfonamido)phenyl)thiophen-2-yl)acrylate(190 mg, yield 82%). Step 4 of Scheme II gave 140 mg of product (yield77%) followed by Step 5 (with purification by prep-HPLC) gave Example 9(41.9 mg, 31% yield). ¹H NMR (DMSO-d₆ 400 MHz): δ 9.76 (s, 1H), 7.82 (d,J=2.0 Hz, 1H), 7.69 (d, J=2.0 Hz, 1H), 7.66 (s, 1H), 7.43 (d, J=8.8 Hz,2H), 7.27 (d, J=8.8 Hz, 2H), 7.14 (d, J=8.4 Hz, 1H), 7.07 (dd, J=2.0 Hz,J=8.0 Hz, 1H), 7.01 (d, J=2.0 Hz, 1H), 2.97 (s, 3H), 2.79-2.68 (m, 2H),2.44-2.38 (m, 5H), 1.78 (s, 3H); MS (ESI): m/z 496.0 [M+1]⁺.

Example 103-(4-(4-chloro-2-methoxyphenyl)-3-(2-methyl-4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoicacid

Followed the procedure described in General Scheme II, withmodification. Step 1 was followed where the starting material wasN-(4-(4-bromo-2-formylthiophen-3-yl)-3-methylphenyl)methanesulfonamide(Intermediate 4) instead of 3,4-dibromothiophene-2-carbaldehyde(Intermediate I), starting material 8A was4-chloro-2-methoxyphenylboronic acid. After purification by columnchromatography (PE:EtOAc=3:1)N-(4-(4-(4-chloro-2-methoxyphenyl)-2-formylthiophen-3-yl)-3-methylphenyl)methanesulfonamide(380 mg, yield 365.2%) was obtained. Step 3 of Scheme II was thenfollowed with column conditions (PE: EtOAc=3:1) to afford (E)-ethyl3-(4-(4-chloro-2-methoxyphenyl)-3-(2-methyl-4-(methylsulfonamido)phenyl)thiophen-2-yl)acrylate(280 mg, yield 63.4%). Step 4 of Scheme II gave 200 mg of product (yield71%) followed by Step 5 (with purification by prep-HPLC) gave Example 10(41.9 mg, 13% yield). ¹H NMR (DMSO-d₆ 400 MHz): δ 9.73 (s, 1H), 7.38 (s,1H), 6.93 (m, 4H), 6.90 (s, 1H), 6.86 (m, 1H), 3.5 (d, 3H), 2.95 (s,1H), 2.75 (m, 2H), 7.01 (t, J=7.2 Hz, 2H), 1.82 (s, 3H); MS (ESI): m/z502.0 [M+23]⁺.

Example 113-(4′-(4-carbamoyl-2-methylphenyl)-5-(2-methyl-1H-imidazol-1-yl)-2,3′-bithiophen-5′-yl)propanoicacid

Followed the procedure described in General Scheme II, withmodification. Followed Scheme II, Step 1 where the starting material 8Awas 4-cyano-2-methylphenylboronic acid to give4-(4-bromo-2-formylthiophen-3-yl)-3-methylbenzonitrile. This wasfollowed by a modification of the second step (tin coupling instead ofboronic acid coupling) which is detailed below.

Step 2: A mixture of Intermediate 5(2-methyl-1-(5-(tributylstannyl)thiophen-2-yl)-1H-imidazole, 580 mg,1.28 mmol), 4-(4-bromo-2-formylthiophen-3-yl)-3-methylbenzonitrile (392mg, 1.28 mmol) and tetrakis(triphenylphosphine)palladium (200 mg, 0.17mmol) in toluene was heated to reflux overnight. The mixture was pouredinto water and extracted with ethyl acetate. The combined organic layerwas concentrated, the residue was purified on silica gel column to give4-(5′-formyl-5-(2-methyl-1H-imidazol-1-yl)-2,3′-bithiophen-4′-yl)-3-methylbenzonitrile(280 mg, yield 56.2%).

Next followed Step 3 of Scheme II to give (E)-ethyl3-(4′-(4-cyano-2-methylphenyl)-5-(2-methyl-1H-imidazol-1-yl)-2,3′-bithiophen-5′-yl)acrylate(120 mg, yield 36.4%). Following Step 4 of Scheme II gave ethyl3-(4′-(4-cyano-2-methylphenyl)-5-(2-methyl-1H-imidazol-1-yl)-2,3′-bithiophen-5′-yl)propanoate.And finally Step 5 of Scheme II was followed (purified directly bypreparative HPLC (26-53% acetonitrile+0.1% trifluoroacetic acid inwater+0.1% trifluoroacetic acid, over 15 min.)) to give Example 11 (55mg, 50% yield). ¹H NMR (CD₃OD 400 MHz): δ 7.82 (m, 2H), 7.72 (s, 1H),7.60 (s, 1H), 7.54 (s, 1H), 7.34 (d, J=7.6 Hz, 1H), 7.16 (d, J=4.0 Hz,1H), 7.85 (d, J=4.0 Hz, 1H), 2.87 (m, 2H), 2.54 (m, 5H), 2.08 (s, 3H);MS (ESI): m/z 452.0 [M+1]⁺.

Example 123-(5-(2-methyl-1H-imidazol-1-yl)-4′-(2-methyl-4-(methylsulfonamido)phenyl)-2,3′-bithiophen-5′-yl)propanoicacid

Prepared following General Scheme II, with modification. Intermediate 4(N-(4-(4-bromo-2-formylthiophen-3-yl)-3-methylphenyl)methanesulfonamide)was coupled with Intermediate 5(2-methyl-1-(5-(tributylstannyl)thiophen-2-yl)-1H-imidazole) followingthe procedure detailed in Example 11, Step 2 to giveN-(4-(5′-formyl-5-(2-methyl-1H-imidazol-1-yl)-2,3′-bithiophen-4′-yl)-3-methylphenyl)methanesulfonamide(200 mg, yield 54.5%). Next followed Step 3 of Scheme II to give(E)-ethyl3-(5-(2-methyl-1H-imidazol-1-yl)-4′-(2-methyl-4-(methylsulfonamido)phenyl)-2,3′-bithiophen-5′-yl)acrylate(180 mg, yield 78.0%). Following Step 4 of Scheme II gave ethyl3-(5-(2-methyl-1H-imidazol-1-yl)-4′-(2-methyl-4-(methylsulfonamido)phenyl)-2,3′-bithiophen-5′-yl)propanoate(160 mg, yield 88.8%). And finally Step 5 of Scheme II was followed(purified directly by preparative HPLC (26-53% acetonitrile+0.1%trifluoroacetic acid in water+0.1% trifluoroacetic acid, over 15 min.))to give Example 12 (15 mg, 10% yield). ¹H NMR (DMSO-d₆ 300 MHz): δ 9.85(s, 1H), 7.80 (s, 1H), 7.38 (s, 1H), 7.10 (m, 5H), 6.83 (d, J=3.9 Hz,1H), 3.02 (s, 3H), 2.74 (m, 2H), 2.44 (m, 2H), 2.27 (s, 3H), 1.91 (s,3H); MS (ESI) m/z: 502.2 [M+1]⁺.

Example 133-(4-(4-bromophenyl)-3-(4-carbamoyl-2-methylphenyl)thiophen-2-yl)propanoicacid

Followed the procedure described in General Scheme II, withmodification. The starting material 8A in Scheme II, Step 1 was4-cyano-2-methylphenylboronic acid to give4-(4-bromo-2-formylthiophen-3-yl)-3-methylbenzonitrile (2.4 g, yield88.9%). The starting material 9A in Step 2 was 4-aminophenylboronicacid, and the crude was purified by column chromatography (PE:EtOAc=5:1) to give4-(4-(4-aminophenyl)-2-formylthiophen-3-yl)-3-methylbenzonitrile (1.0 g,yield 64.0%). Step 3 of Scheme II was followed to give crude (E)-ethyl3-(4-(4-aminophenyl)-3-(4-cyano-2-methylphenyl)thiophen-2-yl)acrylatewhich was used directly in the next step. Following Step 4 of Scheme IIwhere the crude was purified by column chromatography (PE: EtOAc=5:1)gave ethyl3-(4-(4-aminophenyl)-3-(4-cyano-2-methylphenyl)thiophen-2-yl)propanoate(330 mg, yield 30.0%). The following modification of Scheme II was made,converting the amino group to bromo:

Synthesis of ethyl3-(4-(4-bromophenyl)-3-(4-cyano-2-methylphenyl)thiophen-2-yl)propanoate

To a mixture of ethyl3-(4-(4-aminophenyl)-3-(4-cyano-2-methylphenyl)thiophen-2-yl)propanoate(180 mg, 0.46 mmol) in a mixture solvent of HBr (48%, 3 mL) and water (1mL) was added dropwise a solution of NaNO₂ (32 mg, 0.46 mmol) in water(0.5 mL). Then the mixture was stirred at 0° C. for 1 hour. Then CuBr(66 mg, 0.46 mmol) was added and the mixture was stirred at 0° C. foradditional 1 hour. The reaction mixture was extracted with EtOAc and thecombined organic layer was dried over Na₂SO₄, concentrated in vacuo togive crude ethyl3-(4-(4-bromophenyl)-3-(4-cyano-2-methylphenyl)thiophen-2-yl)propanoate,which was used in the next step directly.

Lastly, Step 5 of Scheme II was followed: A mixture of crude ethyl3-(4-(4-bromophenyl)-3-(4-cyano-2-methylphenyl)thiophen-2-yl)propanoate(210 mg, 0.46 mmol) in DMSO (3 mL) was added aqueous solution of NaOH(2M, 1 mL), followed by the addition of H₂O₂ (0.2 mL). The mixture wasstirred at room temperature for 10 minutes. The mixture was quenchedwith saturated aqueous Na₂SO₃ and extracted with a mixture of DCM/iPrOH(3/1). The combined organic layer was dried over Na₂SO₄, concentrated invacuo. The residue was purified by HPLC (48-78% acetonitrile+0.15%trifluoroacetic acid in water, over 15 min.) to afford3-(4-(4-bromophenyl)-3-(4-carbamoyl-2-methylphenyl)thiophen-2-yl)propanoicacid (17.0 mg, yield 8.3%). ¹H NMR (CD₃OD 400 MHz): δ 7.66 (d, J=8.0 Hz,1H), 7.62 (s, 1H), 7.26 (d, J=8.0 Hz, 1H), 7.20-7.19 (m, 3H), 7.11-7.08(m, 2H), 2.62-2.81 (m, 2H), 2.50 (t, J=7.6 Hz, 2H), 1.98 (s, 3H). MS(ESI): m/z 446.0 [M+3]⁺.

Example 143-(4-(4-carbamoyl-2-methylphenyl)-5-(4-methoxyphenyl)thiophen-3-yl)propanoicacid

Step 1 of General Scheme III: Synthesis of4-(4-bromothiophen-3-yl)-3-methylbenzonitrile

A mixture of 3,4-dibromothiophene (3 g, 12.5 mmol),3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (2g, 12.5 mmol), Na₂CO₃ (2.7 g, 25 mmol), Pd(PPh₃)₄ (1.4 g, 1.25 mmol) ina mixture of toluene/EtOH/H₂O (40 mL, 5:3:2) was stirred at 80° C. undernitrogen overnight. The reaction mixture was diluted with EtOAc (80 mL),and washed with water and brine. The organic phase was dried overNa₂SO₄, concentrated in vacuo, and purified on silica gel column (PE:EtOAc=30:1) to afford 4-(4-bromothiophen-3-yl)-3-methylbenzonitrile(Scheme III, 13, X₁=4-cyano-2-methylphenyl) (2.4 g, yield 68%) as yellowoil. ¹H NMR (CD₃OD 400 MHz): δ 7.56 (s, 1H), 7.50-7.48 (m, 2H), 7.31 (d,J=3.2 Hz, 1H), 7.22 (d, J=7.6 Hz, 1H), 2.10 (s, 3H).

Step 2 of General Scheme III: Synthesis of (E)-ethyl3-(4-(4-cyano-2-methylphenyl)thiophen-3-yl)acrylate

To a solution of 4-(4-bromothiophen-3-yl)-3-methylbenzonitrile (2.4 g,8.7 mmol) and ethyl 2-(tritylphosphinylidene)acetate (2.6 g, 26 mmol) inDMF (10 mL) was added Pd(OAc)₂ (194 mg, 0.87 mmol) and PPh₃ (228 mg,0.87 mmol), and the mixture was stirred at 150° C. under nitrogenovernight. The mixture was diluted with EtOAc. The organic layer waswashed with water and brine, dried over Na₂SO₄, concentrated in vacuo,and purified on silica gel column (PE: EtOAc=20:1) to give (E)-ethyl3-(4-(4-cyano-2-methylphenyl)thiophen-3-yl)acrylate (Scheme III, 14,X₁=4-cyano-2-methylphenyl) (1 g, yield 39%) as yellow oil. ¹H NMR (CD₃OD400 MHz): δ 8.03 (d, J=2.8 Hz, 1H), 7.71 (s, 1H), 7.64 (d, J=7.6 Hz,1H), 7.39-7.34 (m, 2H), 7.28 (d, J=16 Hz, 1H), 6.06 (d, J=16.4 Hz, 1H),4.15 (q, J=7.2 Hz, 2H), 2.15 (s, 3H), 1.24 (t, J=7.2 Hz, 3H).

Step 3 of General Scheme III: Synthesis of ethyl3-(4-(4-cyano-2-methylphenyl)thiophen-3-yl)propanoate

To a solution of (E)-ethyl3-(4-(4-cyano-2-methylphenyl)thiophen-3-yl)acrylate (1 g, 3.4 mmol) inEtOH (20 mL) was added 10% Pd/C (w/w, 400 mg), and the mixture wasstirred at room temperature under 1 atm of H₂ overnight. The reactionmixture was filtered, and concentrated to give ethyl3-(4-(4-cyano-2-methylphenyl)thiophen-3-yl)propanoate (Scheme III, 15,X₁=4-cyano-2-methylphenyl) (0.9 g, yield 90%) as gray oil. ¹H NMR (CD₃OD400 MHz): δ 7.69 (s, 1H), 7.60 (dd, J=8 Hz, J=1.2 Hz, 1H), 7.34 (d, J=8Hz, 1H), 7.26-7.23 (m, 2H), 4.05 (q, J=7.2 Hz, 2H), 2.67 (t, J=7.6 Hz,2H), 2.45 (t, J=7.6 Hz, 2H), 2.19 (s, 3H), 1.19 (t, J=7.2 Hz, 3H).

Step 4 of General Scheme III: Synthesis of ethyl3-(5-bromo-4-(4-cyano-2-methylphenyl)thiophen-3-yl)propanoate

To a solution of compound 5 (500 mg, 1.7 mmol) in DMF (10 mL) was addedNBS (296 mg, 1.7 mmol), and the resulting mixture was stirred at 100° C.overnight. When TLC showed the starting material was consumed. Themixture was diluted with EtOAc, washed with water and brine,concentrated, and purified by preparative HPLC to afford the desiredethyl 3-(5-bromo-4-(4-cyano-2-methylphenyl)thiophen-3-yl)propanoate(Scheme III, 16, X₁=4-cyano-2-methylphenyl) (15 mg, yield 2%) (theundesired isomer was isolated in 41% yield). ¹H NMR (DMSO-d₆ 400 MHz):δ7.86 (s, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.43 (s, 1H), 7.30 (d, J=8.0 Hz,1H), 3.96 (q, J=7.2 Hz, 2H), 2.42 (s, 2H), 2.05 (s, 5H), 1.09 (t, J=7.2Hz, 3H).

Step 5 of General Scheme III: Synthesis of3-(4-(4-cyano-2-methylphenyl)-5-(4-methoxyphenyl)thiophen-3-yl)propanoicacid

A mixture of ethyl3-(5-bromo-4-(4-cyano-2-methylphenyl)thiophen-3-yl)propanoate (35 mg,0.09 mmol), 4-methoxyphenylboronic acid (16 mg, 0.10 mmol), Na₂CO₃ (19mg, 0.18 mmol), Pd(PPh₃)₂Cl₂ (6 mg, 0.009 mmol) in a mixture of dioxaneand H₂O (5 mL, 4:1) was stirred at 100° C. overnight under nitrogen. Themixture was neutralized to pH=5, and extracted with EtOAc. The organiclayer was concentrated, and purified by preparative TLC to give3-(4-(4-cyano-2-methylphenyl)-5-(4-methoxyphenyl)thiophen-3-yl)propanoicacid (30 mg, yield 86%) as yellow oil.

Step 6 of General Scheme III: Synthesis of3-(4-(4-carbamoyl-2-methylphenyl)-5-(4-methoxyphenyl)thiophen-3-yl)propanoicacid

To a solution of3-(4-(4-cyano-2-methylphenyl)-5-(4-methoxyphenyl)thiophen-3-yl)propanoicacid (30 mg, 0.08 mmol) in DMSO (5 mL) was added NaOH (6 mg, 0.16 mmol),and 30% H₂O₂ (9 mg, 0.08 mmol). The mixture was stirred at roomtemperature for 2 h, and purified by preparative HPLC to give 16 mg ofthe title compound, Example 14 (Scheme III, 17,X₁=4-carbamoyl-2-methylphenyl, X₂=4-methoxyphenyl) (yield 52%) as awhite solid. ¹H NMR (CD₃OD 400 MHz TMS): δ7.72 (s, 2H), 7.26 (d, J=8.4Hz, 1H), 7.14 (s, 1H), 7.04 (d, J=8.8 Hz, 2H), 6.70 (d, J=8.4 Hz, 2H),3.70 (s, 3H), 2.57 (t, J=7.2 Hz, 2H), 2.38 (t, J=8.0 Hz, 2H), 1.96 (s,3H); MS (ESI): m/z 317.9 [M+23]⁺.

Example 15 Synthesis of Intermediates Referenced in the Above ExamplesIntermediate 1: 3,4-dibromothiophene-2-carbaldehyde

To a solution of diisopropylamine (1.3 g, 12.5 mmol) in 15 mL ofanhydrous THF was added n-BuLi (4.0 mL, 2.5 M in hexane) at −78° C.After the mixture was stirred at −78° C. for 0.5 h 3,4-dibromothiophene(2 g, 8.3 mmol) in anhydrous THF (20 mL) was added. The mixture wasstirred at −78° C. for 15 min., a solution of DMF (670 mg, 9.2 mmol) inTHF (5 mL) was added. After being stirred at this temperature for 0.5 h,the mixture was stirred at room temperature for 2 hours. The mixture wasquenched with NH₄Cl saturated solution, diluted with water, extractedwith EtOAc, concentrated, and purified by column chromatography to give3,4-dibromothiophene-2-carbaldehyde (1.0 g, yield 45%).

Intermediate 2:2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-imidazole

Step 1: A mixture of 1-fluoro-4-nitrobenzene (10 g, 70.9 mmol),2-methyl-1H-imidazole (5.8 g, 70.9 mmol), and Cs₂CO₃ (34.7 g, 106.4mmol) in degassed DMF (200 mL) was heated at 100° C. under nitrogenovernight. When TLC indicated that 1-fluoro-4-nitrobenzene was consumed,the reaction mixture was concentrated in vacuo. The residue was dilutedwith water (300 mL), and a grey precipitate was formed and was isolatedto give 2-methyl-1-(4-nitrophenyl)-1H-imidazole (12.8 g, yield 89%).

Step 2: To a solution 2-methyl-1-(4-nitrophenyl)-1H-imidazole (12.8 g,63 mmol) in MeOH (150 mL) was added Pd/C (3 g). The resulting mixturewas stirred at room temperature for 8 hours under hydrogen. When TLCindicated the starting material was consumed, the mixture was filteredand concentrated in vacuo to give 4-(2-methyl-1H-imidazol-1-yl)aniline(10 g, yield 92%).

Step 3: To a solution of 4-(2-methyl-1H-imidazol-1-yl)aniline (10 g,57.8 mmol) in 100 mL of HBr solution was added a solution of NaNO₂ (5.2g, 75.1 mmol) in H₂O (60 mL) at 0° C. Stirred at this temperature for 20min., then poured the mixture into a solution of CuBr (58 g, 404 mmol)in HBr solution (200 mL) at 0° C., and stirred for 30 min., diluted withwater, and stirred at room temperature for 1 hour. The precipitate wasseparated out, and evaporated in vacuo to give 28 g of crude product,which was purified by Prep-HPLC to obtain1-(4-bromophenyl)-2-methyl-1H-imidazole (7 g, yield 51%).

Step 4: A mixture of 1-(4-bromophenyl)-2-methyl-1H-imidazole (3.6 g,15.3 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane)(4.3 g, 16.8 mmol), KOAc (3.0 g, 30.5 mmol), and Pd(dppf)Cl₂ (559 mg,0.76 mmol) in DMF (40 mL) was stirred at 100° C. overnight. When TLCindicated the starting material was consumed, the mixture wasconcentrated and purified by column chromatography (PE:EtOAc=1:2) toafford2-methyl-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-imidazole(1.1 g, yield 26%).

Intermediate 3: 4-(4-bromo-5-formylthiophen-3-yl)benzonitrile

To a solution of diisopropylamine (1.2 g, 11.4 mmol) in 20 mL ofanhydrous THF was added n-BuLi (3.7 mL, 2.5 M in hexane) at −78° C.After the mixture was stirred at this temperature for 0.5 h, a solutionof 4-(4-bromothiophen-3-yl)benzonitrile (prepared in Step 1 of Compound2) (2.0 g, 7.6 mmol) in anhydrous THF (20 mL) was added dropwise. Themixture was stirred at −78° C. for 15 min., a solution of DMF (611 mg,8.4 mmol) in THF (5 mL) was added. The reaction mixture was stirred at−78° C. for 0.5 h, and at room temperature for 2 h. The mixture wasquenched with saturated NH₄Cl aqueous solution, extracted with EtOAc,concentrated, and purified by column chromatography on silica gel togive 4-(4-bromo-5-formylthiophen-3-yl)benzonitrile (600 mg, yield 27%).

Intermediate 4:N-(4-(4-bromo-2-formylthiophen-3-yl)-3-methylphenyl)methanesulfonamide

Step 1: To the solution of N-(4-bromo-3-methylphenyl)methanesulfonamide(11.5 g, 43 mmol) in DMF were added bis(pinacolato)diboron (12 g, 47.3mmol) and KOAc (8.4 g, 86 mmol), the mixture was degassed with N₂, andPdCl₂(dppf) (2 g, 2.7 mmol) was added, then the mixture was degassedagain, and the resulting mixture was heated to 100° C. under N₂overnight. The reaction solution was diluted with EtOAc (500 mL), andfiltrated by a celite, washed with EtOAc, the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography to giveN-(3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanesulfonamide(8.2 g, yield 60%).

Step 2: To the solution of Intermediate 1 (4 g, 15 mmol) in a mixture ofDME and H₂O (150 mL/50 mL) were addedN-(3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanesulfonamide(3.6 g, 1.65 mmol) and Na₂CO₃ (2.8 g, 3.1 mmol), the mixture wasdegassed with N₂, and Pd(PPh₃)₄ (500 mg) was added, then the mixture washeated to 100° C. under N₂ overnight. The reaction solution wasconcentrated to remove the organic solution, then the aqueous layer wasextracted with EtOAc (100 mL×3). The combined organic layers were washedwith brine, dried over Na₂SO₄, concentrated under reduced pressure, theresidue was purified by column chromatography to giveN-(4-(4-bromo-2-formylthiophen-3-yl)-3-methylphenyl)methanesulfonamide(2.0 g, yield 35.7%).

Intermediate 5:2-methyl-1-(5-(tributylstannyl)thiophen-2-yl)-1H-imidazole

Step 1: To the solution of 2,5-dibromothiophene (20 g, 82.6 mmol) and2-methyl-1H-imidazole (4.1 g, 50 mmol) in DMSO (100 mL) was added CuI(3.14 g, 16.5 mmol), L-proline (0.95 g, 8.3 mmol), K₂CO₃ (22.9 g, 0.165mol), the resultant mixture was heated to 100° C. under N₂ balloonprotection overnight. The reaction mixture was filtrated and thefiltrate was poured into water, extracted with EtOAc (300 mL×5). Thecombined organic layers were washed with brine, dried over Na₂SO₄, andthen filtrated, the filtrate was concentrated under reduced pressure,the residue was purified by column chromatography to give desired1-(5-bromothiophen-2-yl)-2-methyl-1H-imidazole (3.8 g, yield 31.2%).

Step 2: To the solution of1-(5-bromothiophen-2-yl)-2-methyl-1H-imidazole (2 g, 8.3 mmol) in driedTHF was added dropwise n-BuLi (2.5 M in hexane, 4 mL, 10 mmol) at −78°C., the mixture was stirred at −78° C. for 30 min. A solution oftributyltin chloride (4.6 g, 0.96 mmol) in THF was added dropwise, thesolution was stirred at −78° C. for 1 hour, and the reaction solutionwas allowed to warm to room temperature and stirred for 1 hour. Thereaction solution was poured into NH₄Cl aqueous solution, the mixturewas extracted with EtOAc (100 mL×3). The organic layer was washed withbrine, dried over Na₂SO₄, then concentrated under reduced pressure, andthe residue was purified by column chromatography to give2-methyl-1-(5-(tributylstannyl)thiophen-2-yl)-1H-imidazole as a yellowoil (1.2 g, yield 31.8%).

Example 16 GSNOR Assays

Various compounds were tested in vitro for their ability to inhibitGSNOR activity. GSNOR inhibitor compounds Examples 1-14 had an IC₅₀ ofabout <10 μM. GSNOR inhibitor compounds Examples 1, 2, 4, 7-9, 11-13, 14had an IC₅₀ of about less than 1.0 μM.

GSNOR expression and purification is described in Biochemistry 2000, 39,10720-10729.

GSNOR fermentation: Pre-cultures were grown from stabs of a GSNORglycerol stock in 2XYT media containing 100 ug/ml ampicillin after anovernight incubation at 37° C. Cells were then added to fresh 2XYT (4 L)containing ampicillin and grown to an OD (A₆₀₀) of 0.6-0.9 at 37° C.before induction. GSNOR expression was induced with 0.1% arabinose in anovernight incubation at 20° C.

GSNOR Purification: E. coli cell paste was lysed by nitrogen cavitationand the clarified lysate purified by Ni affinity chromatography on anAKTA FPLC (Amersham Pharmacia). The column was eluted in 20 mM Tris pH8.0/250 mM NaCl with a 0-500 mM imidazole gradient. Eluted GSNORfractions containing the Smt-GSNOR fusion were digested overnight withUlp-1 at 4° C. to remove the affinity tag then re-run on the Ni columnunder the same conditions. GSNOR was recovered in the flowthroughfraction and for crystallography is further purified by Q-Sepharose andHeparin flowthrough chromatography in 20 mM Tris pH 8.0, 1 mM DTT, 10 uMZnSO₄.

GSNOR assay: GSNO and enzyme/NADH Solutions are made up fresh each day.The solutions are filtered and allowed to warm to room temperature. GSNOsolution: 100 mM NaPO4 (pH 7.4), 0.480 mM GSNO. 396 μL of GSNO Solutionis added to a cuvette followed by 8 μL of test compound in DMSO (or DMSOonly for full reaction control) and mixed with the pipette tip.Compounds to be tested are made up at a stock concentration of 10 mM in100% DMSO. 2 fold serial dilutions are done in 100% DMSO. 8 μL of eachdilution are added to an assay so that the final concentration of DMSOin the assay is 1%. The concentrations of compounds tested range from100 to 0.003 μM. Enzyme/NADH solution: 100 mM NaPO₄ (pH 7.4), 0.600 mMNADH, 1.0 μg/mL GSNO Reductase. 396 μL of the Enzyme/NADH solution isadded to the cuvette to start the reaction. The cuvette is placed in theCary 3E UV/Visible Spectrophotometer and the change in 340 nmabsorbance/min at 25° C. is recorded for 3 minutes. The assays are donein triplicate for each compound concentration. IC₅₀'s for each compoundare calculated using the standard curve analysis in the Enzyme KineticsModule of SigmaPlot.

Final assay conditions: 100 mM NaPO₄, pH 7.4, 0.240 mM GSNO, 0.300 mMNADH, 0.5 μg/mL GSNO Reductase, and 1% DMSO. Final volume: 800μL/cuvette.

Example 17 Efficacy of GSNORi in Experimental Asthma

Experimental Asthma Model

A mouse model of ovalbumin (OVA)-induced asthma can be used to screenGSNOR inhibitors for efficacy against methacholine (MCh)-inducedbronchoconstriction/airway hyper-responsiveness. This is a widely usedand well characterized model that presents with an acute, allergicasthma phenotype with similarities to human asthma. Efficacy of GSNORinhibitors is assessed using a protocol in which GSNOR inhibitors areadministered after OVA sensitization and airway challenge, and prior tochallenge with MCh. Bronchoconstriction in response to challenge withincreasing doses of MCh is assessed using whole body plethysmography(P_(enh); Buxco). The amount of eosinophil infiltrate into thebronchoaveolar lavage fluid (BALF) is also determined as a measure oflung inflammation. The effect of GSNOR inhibitors is compared tovehicles and to Combivent (inhaled; IH) as the positive control.

Materials and Method

Allergen Sensitization and Challenge Protocol

OVA (500 μg/ml) in PBS is mixed with equal volumes of 10% (w/v) aluminumpotassium sulfate in distilled water and incubated for 60 min. at roomtemperature after adjustment to pH 6.5 using 10 N NaOH. Aftercentrifugation at 750×g for 5 min, the OVA/alum pellet is resuspended tothe original volume in distilled water. Mice received an intraperitoneal(IP) injection of 100 μg OVA (0.2 mL of 500 μg/mL in normal saline)complexed with alum on day 0. Mice are anesthetized by IP injection of a0.2-mL mixture of ketamine and xylazine (0.44 and 6.3 mg/mL,respectively) in normal saline and are placed on a board in the supineposition. Two hundred fifty micrograms (100 μl of a 2.5 mg/ml) of OVA(on day 8) and 125 μg (50 μl of 2.5 mg/ml) OVA (on days 15, 18, and 21)are placed on the back of the tongue of each animal.

Pulmonary Function Testing (Penh)

In vivo airway responsiveness to methacholine is measured 24 h after thelast OVA challenge in conscious, freely moving, spontaneously breathingmice with whole body plethysmography using a Buxco chamber (Wilmington,N.C.). Mice are challenged with aerosolized saline or increasing dosesof methacholine (5, 20, and 50 mg/mL) generated by an ultrasonicnebulizer for 2 min. The degree of bronchoconstriction is expressed asenhanced pause (P_(enh)), a calculated dimensionless value, whichcorrelates with the measurement of airway resistance, impedance, andintrapleural pressure in the same mouse. P_(enh) readings are taken andaveraged for 4 min. after each nebulization challenge. P_(enh) iscalculated as follows: P_(enh)=[(T_(e)/T_(r)−1)×(PEF/PIF)], where T_(e)is expiration time, T_(r) is relaxation time, PEF is peak expiratoryflow, and PIF is peak inspiratory flow ×0.67 coefficient. The time forthe box pressure to change from a maximum to a user-defined percentageof the maximum represents the relaxation time. The T_(r) measurementbegins at the maximum box pressure and ends at 40%.

Eosinophil Infiltrate in BALF

After measurement of airway hyper-reactivity, the mice are exsanguinatedby cardiac puncture, and then BALF is collected from either both lungsor from the right lung after tying off the left lung at the mainstembronchus. Total BALF cells are counted from a 0.05 mL aliquot, and theremaining fluid is centrifuged at 200×g for 10 min at 4° C. Cell pelletsare resuspended in saline containing 10% BSA with smears made on glassslides. Eosinophils are stained for 5 min. with 0.05% aqueous eosin and5% acetone in distilled water, rinsed with distilled water, andcounterstained with 0.07% methylene blue. Alternatively, eosinophils andother leukocytes are stained with DiffQuik.

GSNOR Inhibitors and Controls

GSNOR inhibitors are reconstituted in phosphate buffered saline (PBS),pH 7.4, or 0.5% w/v carboxy methylcellulose at concentrations rangingfrom 0.00005 to 3 mg/mL. GSNOR inhibitors are administered to mice (10mL/kg) as a single dose or multiple dose either intravenously (IV) ororally via gavage. Dosing is performed from 30 min. to 72 h prior to MChchallenge. Effect of GSNOR inhibitors are compared to vehicle dosed inthe same manner.

Combivent is used as the positive control in all studies. Combivent(Boehringer Ingelheim) is administered to the lung using the inhalerdevice supplied with the product, but adapted for administration tomice, using a pipet tip. Combivent is administered 48 h, 24 h, and 1 hprior to MCh challenge. Each puff (or dose) of Combivent provides a doseof 18 μg ipatropium bromide (IpBr) and 103 μg albuterol sulfate orapproximately 0.9 mg/kg IpBr and 5 mg/kg albuterol.

Statistical Analyses

Area under the curve values for P_(enh) across baseline, saline, andincreasing doses of MCh challenge are calculated using GraphPad Prism5.0 (San Diego, Calif.) and expressed as a percent of the respective (IVor orally administered) vehicle control. Statistical differences amongtreatment groups and the respective vehicle control group within eachstudy are calculated using one-way ANOVA, Dunnetts or Bonferronipost-hoc tests or t-test (JMP 8.0, SAS Institute, Cary, N.C. orMicrosoft Excel). A p value of <0.05 among the treatment groups and therespective vehicle control group is considered significantly different.

Example 18 Mouse Pharmacokinetic (PK) Study

Experimental Model

The mouse can be used to determine the pharmacokinetics of compounds ofthe invention. This species is widely used to assess the bioavailabilityof compounds by administering both oral (PO) and intravenous (IV) testarticles. Efficacy of the compounds of the invention is compared byassessing plasma exposure in male BALB/c mice either via IV or POadministration at the times of peak activity.

Materials and Methods

IV Administration of Compounds of the Invention

Compounds of the invention are reconstituted in a phosphate bufferedsaline (PBS)/10% Solutol (HS 15) clear solution resulting in aconcentration of 0.2 mg/mL and administered to mice (2 mg/kg) as asingle IV dose. Animals are dosed via the lateral tail vein. Bloodsamples are collected at designated time points (0.083, 0.25, 0.5, 1, 2,4, 8, 16, 24 hours) by cardiac puncture under isoflurane anesthesia (upto 1 mL blood per animal). The blood is collected into tubes containingLi-Heparin. The blood samples are kept on ice until centrifugationwithin approximately 30 minutes of collection. The plasma is transferredinto labeled polypropylene tubes and frozen at −70° C. until analyzed byLC/MS/MS.

PO Administration of Compounds of the Invention

The compounds of the invention are reconstituted in 40% PropyleneGlycol/40% Propylene Carbonate/20% of a 5% Sucrose clear solutionresulting in a concentration of 2 mg/mL and administered to mice (10mg/kg) as a single oral dose via gavage. Blood samples are collected at0.25, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours post dose by cardiacpuncture under isoflurane anesthesia. The blood is collected in tubescontaining Li-Heparin. The blood samples are kept on ice untilcentrifugation within approximately 30 minutes of collection. The plasmais transferred into labeled polypropylene tubes and frozen at −70° C.until analyzed by LC/MS/MS.

LC/MS/MS Analysis

Plasma samples at each timepoint are analyzed using a LC-MS/MS with alower limit of quantification (LLOQ) of 1 ng/mL. Plasma is analyzed todetermine the amount of the compound of the invention in each sample andregression curves generated for each compounds of the invention in therelevant matrixes.

WinNonlin analysis is used for calculating PK parameters for both the IVand PO administrations:

PK parameters for IV portion—AUC_(iast); AUC_(INF); T1/2; Cl; Vss;C_(max); MRT

PK parameters for PO portion—AUC_(last); AUC_(INF); T1/2; C_(max); Cl,MRT.

In addition to the above PK parameters, bioavailability (% F) can becalculated.

Example 19 Efficacy of GSNOR Inhibitors in Experimental InflammatoryBowel Disease (IBD)

Overview of the Models:

Acute and chronic models of dextran sodium sulfate (DSS)-induced IBD inmice can be used to explore efficacy of GSNORi against this disease.Acute and chronic DSS-induced IBD are widely used and well characterizedmodels that induce pathological changes in the colon similar to thoseobserved in the human disease. In these models and in human disease,epithelial cells within the crypts of the colon are disrupted, leadingto dysfunction of the epithelial barrier and the ensuing tissueinflammation, edema, and ulceration. GSNORi therapy may benefit IBD byrestoring s-nitrosoglutathione (GSNO) levels, and thus prevent orreverse the epithelial barrier dysfunction.

Acute Prophylactic Model:

Experimental IBD is induced by administration of DSS in the drinkingwater of male C57Bl/6 mice (N=8 to 10 mice per group) for 6 consecutivedays. GSNORi is dosed orally at doses of 0.1 to 10 mg/kg/day for 10 daysstarting two days prior to and continuing two days post DSS exposure.Two days post DSS exposure, the effect of GSNORi is assessed in ablinded fashion via endoscopy and histopathology using a five pointscale ranging from a score=0 (normal tissue) through a score=4(ulcerative tissue damage and marked pathological changes). Levels ofcirculating cytokines involved in inflammatory pathways are alsoassessed. The effect of GSNORi is compared to vehicle treated controls.The corticosteroid, prednisolone, is used as the positive control inthis study and is administered daily at 3 mg/kg/day via oral dosing.Naïve mice (N=5) are also assessed as a normal tissue control.

Chronic Treatment Model:

Experimental IBD is induced by administration of DSS in the drinkingwater of male C57Bl/6 mice (N=10 to 12 mice per group) for 6 consecutivedays. GSNORi is dosed orally at doses of 10 mg/kg/day for 14 daysstarting one day after cessation of DSS exposure. Efficacy of GSNORi isassessed in a blinded fashion via endoscopy after 7 days and 14 days ofGSNORi dosing and via histopathology after 14 days of GSNORi dosingusing a five point scale ranging from a score=0 (normal tissue) througha score=4 (ulcerative tissue damage and marked pathological changes).Levels of circulating cytokines involved in inflammatory pathways arealso assessed. The effect of GSNORi is compared to vehicle treatedcontrols. The corticosteroid, prednisolone, is used as the positivecontrol in this study and is administered daily at 3 mg/kg/day via oraldosing. Naïve mice (N=5) are also assessed as a normal tissue control.

Example 20 Efficacy of GSNOR Inhibitors in Experimental ChronicObstructive Pulmonary Disease (COPD)

Short Duration Cigarette Smoke COPD Models

The efficacy of GSNOR inhibitors can be assessed in a mouse model ofchronic obstructive pulmonary disease (COPD) induced by short duration(4 days or 11 days) of exposure to cigarette smoke. Infiltration ofinflammatory cells into the bronchoalveolar lavage fluid (BALF) and BALFlevels of chemokines involved in inflammation and tissue turnover/repairare measured to assess the influences of GSNOR inhibitors on some of theearly events associated with the initiation and progression of COPD.

Overview of the Models:

Efficacy of GSNOR inhibitors against COPD is explored using acute (4day) and subchronic (11 day) models of cigarette smoke-induced COPD inmice. Exposure of animals to cigarette smoke provides a model of COPD inwhich injury is induced by the same causative agent as in human diseaseand in which injury exhibits similarities to the human disease,including airway obstruction, airspace enlargement, and involvement ofinflammatory responses in these pathologies. In animal models, changesin lung pathology are only evident after extended (several months)duration of exposure to cigarette smoke, thus making chronic modelsprohibitive as effective screening tools. More recently, modelsexploring inflammatory responses after short duration (2 weeks or less)of smoke exposure in mice have been utilized as tools for screeningefficacy and mechanisms of action of novel therapeutics against COPD.The key roles of inflammation in the initiation and progression of COPD,make these short duration models relevant for initial tests of efficacyof novel therapeutics.

Acute (4 day) smoke exposure model: Female C57Bl/6 mice (N=8 per group)are exposed to cigarette smoke using a whole body exposure chamber. Miceare exposed daily for 4 consecutive days to 4 cycles of smoke from 6sequential cigarettes (Kentucky 3R4F without filter) with a 30 minutesmoke free interval between cycles. GSNOR inhibitors are administereddaily via oral dosing at 10 mg/kg/day for 7 days starting 2 days priorto smoke exposure and continuing 1 day post-exposure. The effects ofGSNOR inhibitors are assessed by quantization of the numbers of totalcells, leukocytes, and leukocytes differentials in the BALF via lightmicroscopy and the levels of BALF chemokines via ELISA at approximately24 h after the last smoke exposure. The effect of GSNOR inhibitors arecompared to vehicle treated controls. The PDE4 inhibitor, roflumilast,is used as the positive control for the study. A group of naïve mice(N=8) is exposed to air and used as a negative control for the study.

Subchronic (11 day) smoke exposure model: Female C57Bl/6 mice (N=10 pergroup) are exposed to cigarette smoke generated from Marlboro 100cigarettes without filters. Exposure times are 25 min. on study day 1,35 min. on study day 2, and 45 min. on study days 3 to 11. GSNORinhibitors are administered one hour prior to smoke exposure on eachday. GSNOR inhibitors are dosed orally at 1 to 10 mg/kg/day for 11 days.The effects of GSNOR inhibitors are assessed by quantization of thenumber of total cells, and leukocytes differentials in the BALF vialight microscopy at 24 h after the last exposure. The effect of GSNORinhibitors are compared to vehicle treated controls and expressed aspercent inhibition of the cigarette smoke induced increases in BALF cellnumbers. Roflumilast is used as the positive control for the study andis dosed at 5 mg/kg/day. A group of naïve mice (N=10) is exposed to airand dosed with vehicle as a negative control for the study.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention.

What is claimed is:
 1. A method of treatment of a disease or conditioncomprising administering a therapeutically effective amount of acompound Formula I or a pharmaceutically acceptable salt thereof:

wherein: X and Y are selected from the group consisting of S or CH,provided that when X is S, Y must be CH and when X is CH, Y must be S;Ar is selected from the group consisting of phenyl and thiophen-yl; R₁is selected from the group consisting of hydrogen, unsubstitutedimidazolyl, substituted imidazolyl, carbamoyl, chloro, bromo, fluoro,hydroxy, and methoxy; R₂ is selected from the group consisting ofhydrogen, methyl, chloro, fluoro, hydroxy, methoxy, ethoxy, propoxy,carbamoyl, dimethylamino, amino, formamido, and trifluoromethyl; R₃ isselected from the group consisting of hydrogen, methyl, methoxy, chloro,and fluoro; and R₄ is selected from the group consisting of CONH₂,NHSO₂CH₃, hydroxy, chloro, and substituted and unsubstituted imidazolyl.2. The method of claim 1 wherein Ar is selected from the groupconsisting of


3. The method of claim 2 wherein R₁ is

wherein R₅ is selected from the group consisting of hydrogen, methyl andethyl.
 4. The method of claim 2 wherein ArR₁R₂ is selected from thegroup consisting of 4-methoxyphenyl, 4-chloro-2-methoxyphenyl,4-hydroxyphenyl, 4-carbamoylphenyl, and 4-bromophenyl.
 5. The method ofclaim 1 wherein R₃ is selected from the group consisting of hydrogen,methyl, and methoxy.
 6. The method of claim 1 wherein the compound is acompound of Formula II:

wherein: Ar is selected from the group consisting of phenyl andthiophen-yl; R₁ is selected from the group consisting of hydrogen,unsubstituted imidazolyl, substituted imidazolyl, carbamoyl, chloro,bromo, fluoro, hydroxy, and methoxy; R₂ is selected from the groupconsisting of hydrogen, methyl, chloro, fluoro, hydroxy, methoxy,ethoxy, propoxy, carbamoyl, dimethylamino, amino, formamido, andtrifluoromethyl; and R₃ is selected from the group consisting ofhydrogen, methyl, methoxy, chloro, and fluoro; and R₄ is selected fromthe group consisting of CONH₂, NHSO₂CH₃, hydroxy, chloro, andsubstituted and unsubstituted imidazolyl.
 7. The method of claim 6wherein Ar is selected from the group consisting of


8. The method of claim 7 wherein R₁ is

wherein R₅ is selected from the group consisting of hydrogen, methyl andethyl.
 9. The method of claim 7 wherein ArR₁R₂ is selected from thegroup consisting of 4-methoxyphenyl, 4-chloro-2-methoxyphenyl,4-hydroxyphenyl, 4-carbamoylphenyl, and 4-bromophenyl.
 10. The method ofclaim 6 wherein R₃ is selected from the group consisting of hydrogen,methyl, and methoxy.
 11. The method of claim 6 selected from the groupconsisting of3-(3-(4-carbamoyl-2-methylphenyl)-4-(4-methoxyphenyl)thiophen-2-yl)propanoicacid;3-(3-(4-carbamoylphenyl)-4-(4-chloro-2-methoxyphenyl)thiophen-2-yl)propanoicacid;3-(3-(4-chloro-2-methoxyphenyl)-4-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid;3-(4-(4-chloro-2-methoxyphenyl)-3-(4-hydroxyphenyl)thiophen-2-yl)propanoicacid;3-(4-(4-chloro-2-methoxyphenyl)-3-(4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoicacid;3-(4-(4-carbamoylphenyl)-3-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoicacid;3-(3-(4-carbamoyl-2-methylphenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoicacid;3-(3-(4-carbamoylphenyl)-4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)thiophen-2-yl)propanoicacid;3-(4-(4-(2-methyl-1H-imidazol-1-yl)phenyl)-3-(2-methyl-4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoicacid;3-(4-(4-chloro-2-methoxyphenyl)-3-(2-methyl-4-(methylsulfonamido)phenyl)thiophen-2-yl)propanoicacid;3-(4′-(4-carbamoyl-2-methylphenyl)-5-(2-methyl-1H-imidazol-1-yl)-2,3′-bithiophen-5′-yl)propanoicacid;3-(5-(2-methyl-1H-imidazol-1-yl)-4′-(2-methyl-4-(methylsulfonamido)phenyl)-2,3′-bithiophen-5′-yl)propanoicacid; and3-(4-(4-bromophenyl)-3-(4-carbamoyl-2-methylphenyl)thiophen-2-yl)propanoicacid.
 12. The method of claim 1 wherein the compound is a compound ofFormula III:

wherein: Ar is selected from the group consisting of phenyl andthiophen-yl; R₁ is selected from the group consisting of hydrogen,unsubstituted imidazolyl, substituted imidazolyl, carbamoyl, chloro,bromo, fluoro, hydroxy, and methoxy; R₂ is selected from the groupconsisting of hydrogen, methyl, chloro, fluoro, hydroxy, methoxy,ethoxy, propoxy, carbamoyl, dimethylamino, amino, formamido, andtrifluoromethyl; R₃ is selected from the group consisting of hydrogen,methyl, methoxy, chloro, and fluoro; and R₄ is selected from the groupconsisting of CONH₂, NHSO₂CH₃, hydroxy, chloro, and substituted andunsubstituted imidazolyl.
 13. The method of claim 12 wherein Ar isselected from the group consisting of


14. The method of claim 13 wherein R₁ is

wherein R₅ is selected from the group consisting of hydrogen, methyl andethyl.
 15. The method of claim 13 wherein ArR₁R₂ is selected from thegroup consisting of 4-methoxyphenyl, 4-chloro-2-methoxyphenyl,4-hydroxyphenyl, 4-carbamoylphenyl, and 4-bromophenyl.
 16. The method ofclaim 12 wherein R₃ is selected from the group consisting of hydrogen,methyl, and methoxy.
 17. The method of claim 12 selected from the groupconsisting of3-(4-(4-carbamoyl-2-methylphenyl)-5-(4-methoxyphenyl)thiophen-3-yl)propanoicacid.
 18. The method of claim 1 comprising a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of Formula Iaccording to claim 1 together with a pharmaceutically accepted carrieror excipient.
 19. The method of claim 1 wherein said disease orcondition is selected from the group consisting of asthma, chronicobstructive pulmonary disease (COPD), and cystic fibrosis.
 20. A methodof making a pharmaceutical composition according to claim 18 comprisingthe step of combining a compound of formula I

wherein: X and Y are selected from the group consisting of S or CH,provided that when X is S, Y must be CH and when X is CH, Y must be S;Ar is selected from the group consisting of phenyl and thiophen-yl; R₁is selected from the group consisting of hydrogen, unsubstitutedimidazolyl, substituted imidazolyl, carbamoyl, chloro, bromo, fluoro,hydroxy, and methoxy; R₂ is selected from the group consisting ofhydrogen, methyl, chloro, fluoro, hydroxy, methoxy, ethoxy, propoxy,carbamoyl, dimethylamino, amino, formamido, and trifluoromethyl; R₃ isselected from the group consisting of hydrogen, methyl, methoxy, chloro,and fluoro; and R₄ is selected from the group consisting of CONH₂,NHSO₂CH₃, hydroxy, chloro, and substituted and unsubstituted imidazolyl;with a pharmaceutically accepted carrier or excipient.